Diagnostic Assay For Source of Inflammation

ABSTRACT

A method of diagnosing the source of local, acute inflammation has been developed based on the discovery that white cells have different patterns of gene expression, and therefore protein markers, depending on the origin of the inflammation. These differences can be readily accessed by analysis of the white cells obtained at a site to be analyzed, for example, in the synovial fluid of a knee. The analysis, by comparison with the analysis of white cells present in known conditions, can be used to differentiate between inflammation due to bacterial infection, arthritis or gout, for example. The examples demonstrate differential gene expression in cells present in synovial fluid biopsies from patients with confirmed bacterial infection as compared to patients with aseptic loosening or patients with inflammation due to gout.

This claims priority under 35 U.S.C. 119 to U.S. Ser. No. 60/603,313filed Aug. 20, 2004.

BACKGROUND OF THE INVENTION

The present invention is generally in the field of diagnostics fordifferent types of inflammation, for example, whether the inflammationis due to bacterial infection or autoimmune disease.

The immune system is a bodywide network of cells and organs that hasevolved to defend the body against attacks by “foreign” invaders. Theproper targets of the immune system are infectious organisms—bacteriasuch as streptococci; Fungi; Parasites, including the microbes thatcause schistosomiasis; and viruses such as herpes virus.

The lymphoid organs are concerned with the growth, development, anddeployment of lymphocytes, which are white blood cells that are keyoperatives of the immune system. The organs of the immune system areconnected with one another and with other organs of the body by anetwork of lymphatic vessels similar to blood vessels. Immune cells andforeign particles are conveyed through the lymphatic vessels in lymph, aclear fluid that bathes the body's tissues. Cells destined to becomeimmune cells, like all blood cells, arise in the bone marrow fromso-called stem cells. Some develop into myeloid cells, a group typifiedby the large, cell- and particle-devouring white blood cells known asphagocytes; phagocytes include monocytes, macrophages, and neutrophils.Other myeloid descendants become granule-containing inflammatory cellssuch as eosinophils and basophils. Lymphoid precursors develop into thesmall white blood cells called lymphocytes. The two major classes oflymphocytes are B cells and T cells. B cells make antibodies. At leasttwo types of lymphocytes are killer cells—cytotoxic T cells and naturalkiller cells. To attack, cytotoxic T cells need to recognize a specificantigen, whereas natural killer or NK cells do not. Both types containgranules filled with potent chemicals, and both types kill on contact.The killer binds to its target, aims its weapons, and delivers a burstof lethal chemicals. Phagocytes are large white cells that can engulfand digest foreign invaders. They include monocytes, which circulate inthe blood, and macrophages, which are found in tissues throughout thebody, as well as neutrophils, cells that circulate in the blood but moveinto tissues where they are needed. Macrophages are versatile cells;they act as scavengers, they secrete a wide variety of powerfulchemicals, and they play an essential role in activating T cells.Neutrophils are not only phagocytes but also granulocytes: they containgranules filled with potent chemicals. These chemicals, in addition todestroying microorganisms, play a key role in acute inflammatoryreactions. Other types of granulocytes are eosinophils and basophils.Mast cells are granule-containing cells in tissue.

When the immune system malfunctions, it can unleash a torrent ofdisorders and diseases. One of the most familiar is allergy. Allergiessuch as hay fever and hives are related to the antibody known as IgE.Sometimes the immune system's recognition apparatus breaks down, and thebody begins to manufacture antibodies and T cells directed against thebody's own cells and organs. Such cells and autoantibodies, as they areknown, contribute to many diseases. For instance, T cells that attackpancreas cells contribute to diabetes, while an autoantibody known asrheumatoid factor is common in persons with rheumatoid arthritis.

Other types of inflammation may arise due to infection or damage totissue due to trauma or excessive wear. Since treatments differ based onthe origin of the disease or disorder, it is important to know what iseliciting the inflammation.

It is therefore an object of the present invention to provide a methodand materials for rapid diagnosis of the source of inflammation.

SUMMARY OF THE INVENTION

A method of diagnosing the source of local, acute inflammation has beendeveloped based on the discovery that white cells have differentpatterns of gene expression, and therefore different protein markers,depending on the origin of the inflammation. These differences can bereadily accessed by analysis of the white cells obtained at a site to beanalyzed, for example, in the synovial fluid of a knee. The analysis, bycomparison with the analysis of white cells present in known conditions,can be used to differentiate between inflammation due to bacterialinfection, arthritis or gout, for example. The method can also be usedin drug screening, where changes in the expression patterns of knowndiseases or disorders to appear more normal in response to a particulartreatment or drug is indicative of potential efficacy.

The'examples demonstrate differential gene expression in cells presentin synovial fluid biopsies from patients with confirmed bacterialinfection as compared to patients with aseptic loosening or patientswith inflammation due to gout.

DETAILED DESCRIPTION OF THE INVENTION

One of the hallmarks of inflammation is an influx of white blood cellsinto the injured area. For example, acute inflammation in kneeinfections, rheumatoid arthritis, Lyme disease, and gout all involve theparticipation of white blood cells. Since the acute cellular infiltratehas been historically considered to be a stereotyped response, there hasbeen little attention given to studying these cells for diagnosticpurposes. A few in vitro studies have suggested that monocytes,dendritic cells, and neutrophils have the ability to alter their geneexpression depending on the source of inflammation. Using thisinformation, it was postulated that the cells in an inflamed knee,despite appearing the same in different forms of inflammation, may havedifferent and diagnostic gene expression profiles. This was demonstratedin the following examples comparing results in cells present in synovialfluid biopsies from patients with confirmed bacterial infection ascompared to patients with aseptic loosening or patients withinflammation due to gout.

I. Samples to be Analyzed

Samples can be obtained for testing using standard techniques. Typicallysamples are obtained by biopsy or aspiration, for example, of tissue ata site to be analyzed, or of synovial joint fluid. Fluids commonlyaspirated for the evaluation of acute inflammation include synovialfluid, sputum, urine, cerebrospinal fluid, peritoneal lavage fluid,pleural effusion, pericardial effusion, and abscesses among others.Tissues commonly biopsied for the analysis of acute inflammation includeconnective tissues such as bone, muscle, and synovium, solid organs suchas liver, heart, kidney, and brain, and reactive tissues such asperiprosthetic tissues.

Nucleic acid samples used in the methods and assays may be prepared byany available method or process. Methods of isolating total mRNA arealso well known to those of skill in the art. For example, methods ofisolation and purification of nucleic acids are described in detail inChapter 3 of Laboratory Techniques in Biochemistry and MolecularBiology: Hybridization With Nucleic Acid Probes, Part I Theory andNucleic Acid Preparation, Tijssen, (1993) (editor) Elsevier Press. Suchsamples include RNA samples, cDNA synthesized from an mRNA sampleisolated from a cell or tissue of interest, DNA amplified from the cDNA,and RNA transcribed from the amplified DNA. One of skill in the artwould appreciate that it may be desirable to inhibit or destroy RNasepresent in homogenates before homogenates are analyzed.

As described in example 1, a method was developed to stabilize, isolate,and purify RNA from inflamed synovial fluid, as described in thefollowing examples.

Biological samples may be of any biological tissue or fluid containingleukocytes. Frequently the sample will be a “clinical sample” which is asample derived from a patient. Typical clinical samples include, but arenot limited to, synovial fluid, sputum, blood, blood-cells (e.g., whitecells), tissue or fine needle biopsy samples, urine, peritoneal fluid,cerebrospinal fluid, abscesses, and pleural fluid, or cells therefrom.Biological samples may also include sections of tissues, such as frozensections or formalin fixed sections taken for histological purposes.Peroprosthetic tissues are often analyzed for evidence of infection.

Controls may either be normal (i.e., not infected or not-inflammedtissue, for example) or samples from known types or stages of infectionor inflammation or other disease. As described below, comparison of theexpression patterns of the sample to be tested with those of thecontrols can be used to diagnose the disease.

II. Methods of Analysis

Analysis for the purpose of monitoring differential gene expression maybe focused on a variety of tissues and fluids, and may also be used todetect or measure a number of different molecular targets. When a cellexpresses a gene, it transcribes the appropriate RNA, which isultimately translated into a protein. The relevant protein may then belocalized to a variety of intracellular or extracellular locations.

Methods of detecting or measuring gene expression may utilize methodsthat focus on cellular components (cellular examination), or methodsthat focus on examining extracellular components (fluid examination).Because gene expression involves the ordered production of a number ofdifferent molecules, a cellular or fluid examination may be used todetect or measure a variety of molecules including RNA, protein, and anumber of molecules that may be modified as a result of the protein'sfunction. Typical diagnostic methods focusing on nucleic acids includeamplification techniques such as PCR and RT-PCR (including quantitativevariants), and hybridization techniques such as in situ hybridization,microarrays, blots, and others. Typical diagnostic methods focusing onproteins include binding techniques such as ELBA, immunohistochemistry,microarray and functional techniques such as enzymatic assays.

Many biological functions are accomplished by altering the expression ofvarious genes through transcriptional (e.g., through control ofinitiation, provision of RNA precursors, RNA processing, etc.) and/ortranslational control. For example, fundamental biological processessuch as cell cycle, cell differentiation and cell death, are oftencharacterized by variations in the expression levels of groups of genes.Changes in gene expression are also associated with pathogenesis. Thus,changes in the expression levels of particular genes serve as signpostsfor the presence and progression of various diseases or inflammation. Asdescribed herein, it is the differences in expression that are used todetermine the origin of the inflammation—whether it is infection oraseptic inflammation, and if infection, whether it is viral, bacterial,or parasitic in origin. If the source is aseptic inflammation, one coulddetermine the specific underlying disease process, as many autoimmunediseases that are associated with local inflammation. This isparticularly important in certain clinical scenarios when pathogendetection is difficult and gross cellular examination is uninformative.The testing of expression of genes in the leukocytes at the site is mucheasier.

Monitoring changes in gene expression may also provide certainadvantages during drug screening development. By determining whatpatterns of expression are associated with infection as compared toinflammation, one can then test for the effect of a drug, and whethertreatment with a drug, or a particular dosage or treatment schedule iseffective in normalizing the expression pattern.

Monitoring changes in gene expression may also provide informationregarding a patient's susceptibility to disease and probability ofrecovering. This is especially important when treating patients withlocal infections and/or autoimmune diseases.

DEFINITIONS

In the description that follows, numerous terms and phrases known tothose skilled in the art are used. In the interest of clarity andconsistency of interpretation, the definitions of certain terms andphrases are provided.

As used herein, the phrase “detecting the level of expression” includesmethods that quantify expression levels as well as methods thatdetermine whether a gene of interest is expressed at all. Thus, an assaywhich provides a yes or no result without necessarily providingquantification of an amount of expression is an assay that requires“detecting the level of expression” as that phrase is used herein. Asused herein, it is the pattern of expression in addition to theindividual expression that is quantitated or qualified for analysis.

As used herein, oligonucleotide sequences that are complementary to oneor more of the genes described herein refers to oligonucleotides thatare capable of hybridizing under stringent conditions to at least partof the nucleotide sequence of the genes. Such hybridizableoligonucleotides will typically exhibit at least about 75% sequenceidentity at the nucleotide level to the genes, preferably about 80% or85% sequence identity or more preferably about 90% or 95% or moresequence identity to the genes.

“Bind(s) substantially” refers to complementary hybridization between aprobe nucleic acid and a target nucleic acid and embraces minormismatches that can be accommodated by reducing the stringency of thehybridization media to achieve the desired detection of the targetpolynucleotide sequence.

The terms “background” or “background signal intensity” refer tohybridization signals resulting from non-specific binding, or otherinteractions, between the labeled target nucleic acids and components ofthe oligonucleotide array (e.g. the oligonucleotide probes, controlprobes, the array substrate, etc.). Background signals may also beproduced by intrinsic fluorescence of the array components themselves. Asingle background signal can be calculated for the entire array, or adifferent background signal may be calculated for each target nucleicacid. In a preferred embodiment, background is calculated as the averagehybridization signal intensity for the lowest 5% to 10% of the probes inthe army, or, where a different background signal is calculated for eachtarget gene, for the lowest 5% to 10% of the probes for each gene. Oneof skill in the art will appreciate that where the probes to aparticular gene hybridize well and thus appear to be specificallybinding to a target sequence, they should not be used in a backgroundsignal calculation. Alternatively, background may be calculated as theaverage hybridization signal intensity produced by hybridization toprobes that are not complementary to any sequence found in the sample(e.g., probes directed to nucleic acids of the opposite sense or togenes not found in the sample such as bacterial genes where the sampleis mammalian nucleic acids). Background can also be calculated as theaverage signal intensity produced by regions of the array that lack anyprobes at all.

The phrase “hybridizing specifically to” refers to the binding,duplexing or hybridizing of a molecule substantially to or only to aparticular nucleotide sequence or sequences under stringent conditionswhen that sequence is present in a complex mixture (e.g., totalcellular) DNA or RNA.

Assays and methods may utilize available formats to simultaneouslyscreen at least about 100, 1000, 10,000 or about 1,000,000 or moredifferent nucleic acid hybridizations.

The terms “mismatch control” or “mismatch probe” refer to a probe whosesequence is deliberately selected not to be perfectly complementary to aparticular target sequence. For each mismatch (MM) control in ahigh-density array there typically exists a corresponding perfect match(PM) probe that is perfectly complementary to the same particular targetsequence. The mismatch may comprise one or more bases. While themismatch(s) may be located anywhere in the mismatch probe, terminalmismatches are less desirable as a terminal mismatch is less likely toprevent hybridization of the target sequence. In a particularlypreferred embodiment, the mismatch is located at or near the center ofthe probe such that the mismatch is most likely to destabilize theduplex with the target sequence under the test hybridization conditions.

The term “perfect match probe” refers to a probe that has a sequencethat is perfectly complementary to a particular target sequence. Thetest probe is typically perfectly complementary to a portion(subsequence) of the target sequence. The perfect match (PM) probe canbe a “test probe”, a “normalization control” probe, an expression levelcontrol probe and the like. A perfect match control or perfect matchprobe is, however, distinguished from a “mismatch control” or “mismatchprobe.”

As used herein a “probe” is defined as a nucleic acid, preferably anoligonucleotide, capable of binding to a target nucleic acid ofcomplementary sequence through one or more types of chemical bonds,usually through complementary base pairing, usually through hydrogenbond formation. As used herein, a probe may include natural (i.e., A, G,U, C or T) or modified bases (7-deazaguanosine, inosine, etc.). Inaddition, the bases in probes may be joined by a linkage other than aphosphodiester bond, so long as it does not interfere withhybridization. Thus, probes may be peptide nucleic acids in which theconstituent bases are joined by peptide bonds rather than phosphodiesterlinkages.

The term “stringent conditions” refers to conditions under which a probewill hybridize to its target subsequence, but with only insubstantialhybridization to other sequences or to other sequences such that thedifference may be identified. Stringent conditions aresequence-dependent and will be different in different circumstances.Longer sequences hybridize specifically at higher temperatures.Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength and pH.

Typically, stringent conditions will be those in which the saltconcentration is at least about 0.01 to 1.0M sodium ion concentration(or other salts) at pH 7.0 to 8.3 and the temperature is at least about30° C. for short probes (e.g., 10 to 50 nucleotides). Stringentconditions may also be achieved with the addition of destabilizingagents such as formamide.

The “percentage of sequence identity” or “sequence identity” isdetermined by comparing two optimally aligned sequences or subsequencesover a comparison window or span, wherein the portion of thepolynucleotide sequence in the comparison window may optionally compriseadditions or deletions (i.e., gaps) as compared to the referencesequence (which does not comprise additions or deletions) for optimalalignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical subunit(e.g., nucleic acid base or amino acid residue) occurs in both sequencesto yield the number of matched positions, dividing the number of matchedpositions by the total number of positions in the window of comparisonand multiplying the result by 100 to yield the percentage of sequenceidentity. Percentage sequence identity when calculated using theprograms GAP or BESTFIT is calculated using default gap weights.

Homology or identity may be determined by BLAST (Basic Local AlignmentSearch Tool) analysis using the algorithm employed by the programsblastp, blastn, blastx, tblastn and tblastx (Karlin et al., Proc. Natl.Acad. Sci. USA 87, 2264-2268 (1990) and Altschul, J. Mol. Evol. 36,290-300 (1993), fully incorporated by reference) which are tailored forsequence similarity searching. The approach used by the BLAST program isto first consider similar segments between a query sequence and adatabase sequence, then to evaluate the statistical significance of allmatches that are identified and finally to summarize only those matcheswhich satisfy a preselected threshold of significance. For a discussionof basic issues in similarity searching of sequence databases, seeAltschul et al., Nature Genet. 6, 119-129 (1994). The search parametersfor histogram, descriptions, alignments, expect (i.e., the statisticalsignificance threshold for reporting matches against databasesequences), cutoff, matrix and filter are at the default settings. Thedefault scoring matrix used by blastp, blastx, tblasta, and tblastx isthe BLOSUM62 matrix (Henikoff et al., Proc. Natl. Acad. Sci. USA 89,10915-10919 (1992). Four blastn parameters were adjusted as follows Q=10(gap creation penalty); R=10 (gap extension penalty); wink=1 (generatesword hits at every winks position along the query); and gapw=16 (setsthe window width within which gapped alignments are generated). Theequivalent Blastp parameter settings were Q=9; R=2; wink=1; and gapw32.A Bestfit comparison between sequences, available in the GCG packageversion 10.0, uses DNA parameters GAP=50 (gap creation penalty) andLENz=3 (gap extension penalty) and the equivalent settings in proteincomparisons are GAP=8 and LEN=2.

For a particular interrogation of two conditions or sources, it isdesirable to select those genes that display a great difference in theexpression pattern between the two conditions or sources. At least atwo-fold difference is desirable, but a three, five-fold or ten-folddifference may be preferred. Interrogations of the genes or proteins canbe performed to yield information on gene expression as well as on thelevels of the encoded proteins.

Comparison of the expression data, as well as available sequence orother information may be done by researcher or diagnostician or may bedone with the aid of a computer and databases as described herein.

Assay Formats

The genes identified as being differentially expressed may be assessedin a variety of nucleic acid detection assays to detect or quantify theexpression level of a gene or multiple genes in a given sample. Forexample, traditional Northern blotting, nuclease protection, RT-PCR,microarray, and differential display methods may be used for detectinggene expression levels. Methods for assaying for mRNA include Northernblots, slot blots, dot blots, and hybridization to an ordered array ofoligonucleotides. Any method for specifically and quantitativelymeasuring a specific protein or mRNA or DNA product can be used.However, methods and assays are most efficiently designed with array orchip hybridization-based methods for detecting the expression of a largenumber of genes. Any hybridization assay format may be used, includingsolution-based and solid support-based assay formats.

The protein products of the genes identified herein can also be assayedto determine the amount of expression. Methods for assaying for aprotein include Western blot, immunoprecipitation, and radioimmunoassay.The proteins analyzed may be localized intracellularly (most commonly anapplication of immunohistochemistry) or extracellularly (most commonlyan application of immunoassays such as ELISA).

In another format, the relative amounts of proteins produced in a cellpopulation may be analyzed for purposes of diagnosis or the protein fromcellular population that has been exposed to an agent to be tested maybe compared to the amount produced in an unexposed control cellpopulation. In this format, probes such as specific antibodies are usedto monitor the differential expression of the protein in the differentcell populations. Cell lines or populations are exposed to the agent tobe tested under appropriate conditions and time. Cellular lysates may beprepared from the exposed cell line or population and a control,unexposed cell line or population. The cellular lysates are thenanalyzed with the probe, such as a specific antibody.

As noted above, the genes may be assayed in any convenient form. Forexample, they may be assayed in the form mRNA or reverse transcribedmRNA. The genes may be cloned or not and the genes may be amplified ornot. The cloning itself does not appear to bias the representation ofgenes within a population. However, it may be preferable to usepolyA+RNA as a source, as it can be used with less processing steps. Insome embodiments, it may be preferable to assay the protein or peptideencoded by the gene.

The sequences of many of the expression marker genes are in the publicdatabases such as GenBank. The sequences of the genes in GenBank arepublicly available at, for example, www.ncbi.nih.gov. IMAGE gives theclone number from the IMAGE consortium. Information on the genes in theAffymetrix® arrays can also be obtained from Affymetrix®.

One of skill in the art will appreciate that an enormous number of arraydesigns are suitable. The high density array will typically include anumber of probes that specifically hybridize to the sequences ofinterest. See WO 99/32660 for methods of producing probes for a givengene or genes. In a preferred embodiment, the array will include one ormore control probes.

High density array chips include “test probes.” Test probes may beoligonucleotides that range from about 5 to about 500 or about 5 toabout 50 nucleotides, more preferably from about 10 to about 40nucleotides and most preferably from about 15 to about 40 nucleotides inlength. In other particularly preferred embodiments, the probes areabout 20 or 25 nucleotides in length. In another preferred embodiment,test probes are double or single strand DNA sequences. DNA sequences maybe isolated or cloned from natural sources or amplified from naturalsources using natural nucleic acid as templates. These probes havesequences complementary to particular subsequences of the genes whoseexpression they are designed to detect. Thus, the test probes arecapable of specifically hybridizing to the target nucleic acid they areto detect.

In addition to test probes that bind the target nucleic acid(s) ofinterest, the high density array can contain a number of control probes.The control probes fall into three categories referred to herein asnormalization controls; expression level controls; and mismatchcontrols. Normalization controls are oligonucleotide or other nucleicacid probes that are complementary to labeled reference oligonucleotidesor other nucleic acid sequences that are added to the nucleic acidsample. The signals obtained from the normalization controls afterhybridization provide a control for variations in hybridizationconditions, label intensity, “reading” efficiency and other factors thatmay cause the signal of a perfect hybridization to vary between arrays.In a preferred embodiment, signals (e.g. fluorescence intensity) readfrom all other probes in the array are divided by the signal (,fluorescence intensity) from the control probes thereby normalizing themeasurements. Virtually any probe may serve as a normalization control.However, it is recognized that hybridization efficiency varies with basecomposition and probe length. Preferred normalization probes areselected to reflect the average length of the other probes present inthe array; however, they can be selected to cover a range of lengths.The normalization control(s) can also be selected to reflect the(average) base composition of the other probes in the array, however ina preferred embodiment, only one or a few probes are used and they areselected such that they hybridize well (i.e., no secondary structure)and do not match any target-specific probes. Expression level controlsare probes that hybridize specifically with constitutively expressedgenes in the biological sample. Virtually any constitutively expressedgene provides a suitable target for expression level controls. Typicalexpression level control probes have sequences complementary tosubsequences of constitutively expressed “housekeeping genes” includingthe .beta.-actin gene, the transferrin receptor gene, and the GAPDHgene. Mismatch controls may also be provided for the probes to thetarget genes, for expression level controls or for normalizationcontrols. Mismatch controls are oligonucleotide probes or other nucleicacid probes identical to their corresponding test or control probesexcept for the presence of one or more mismatched bases. A mismatchedbase is a base selected so that it is not complementary to thecorresponding base in the target sequence to which the probe wouldotherwise specifically hybridize. One or more mismatches are selectedsuch that under appropriate hybridization conditions (e.g., stringentconditions) the test or control probe would be expected to hybridizewith its target sequence, but the mismatch probe would not hybridize (orwould hybridize to a significantly lesser extent). Preferred mismatchprobes contain a central mismatch. Thus, for example, where a probe is atwenty-mer, a corresponding mismatch probe may have the identicalsequence except for a single base mismatch (e.g., substituting a G, a Cor a T for an A) at any of positions 6 through 14 (the centralmismatch). Mismatch probes thus provide a control for non-specificbinding or cross hybridization to a nucleic acid in the sample otherthan the target to which the probe is directed. Mismatch probes alsoindicate whether hybridization is specific or not.

Solid Supports

Solid supports containing oligonucleotide probes for differentiallyexpressed genes can be any solid or semisolid support material known tothose skilled in the art. Suitable examples include, but are not limitedto, membranes, filters, tissue culture dishes, polyvinyl chloridedishes, beads, test strips, silicon or glass based chips and the like.Suitable glass wafers and hybridization methods are widely available.Any solid surface to which oligonucleotides can be bound, eitherdirectly or indirectly, either covalently or non-covalently, can beused. In some embodiments, it may be desirable to attach someoligonucleotides covalently and others non-covalently to the same solidsupport. A preferred solid support is a high density array or DNA chip.These contain a particular oligonucleotide probe in a predeterminedlocation on the array. Each predetermined location may contain more thanone molecule of the probe, but each molecule within the predeterminedlocation has an identical sequence. Such predetermined locations aretermed features. There may be, for example, from 2, 10, 100, 1000 to10,000, 100,000 or 400,000 of such features on a single solid support.The solid support or the area within which the probes are attached maybe on the order of a square centimeter. Methods of forming high densityarrays of oligonucleotides with a minimal number of synthetic steps areknown. The oligonucleotide analogue array can be synthesized on a solidsubstrate by a variety of methods, including, but not limited to,light-directed chemical coupling, and mechanically directed coupling(see U.S. Pat. No. 5,143,854 to Pirrung et al.; U.S. Pat. No. 5,800,992to Fodor et al.; U.S. Pat. No. 5,837,832 to Chee et al.

In brief, the light-directed combinatorial synthesis of oligonucleotidearrays on a glass surface proceeds using automated phosphoramiditechemistry and chip masking techniques. In one specific implementation, aglass surface is derivatized with a silane reagent containing afunctional group, e.g., a hydroxyl or amine group blocked by aphotolabile protecting group. Photolysis through a photolithographicmask is used selectively to expose functional groups which are thenready to react with incoming 5′ photoprotected nucleosidephosphoramidites. The phosphoramidites react only with those sites whichare illuminated (and thus exposed by removal of the photolabile blockinggroup). Thus, the phosphoramidites only add to those areas selectivelyexposed from the preceding step. These steps are repeated until thedesired array of sequences has been synthesized on the solid surface.Combinatorial synthesis of different oligonucleotide analogues atdifferent locations on the array is determined by the pattern ofillumination during synthesis and the order of addition of couplingreagents.

In addition to the foregoing, methods which can be used to generate anarray of oligonucleotides on a single substrate are described in WO93/09668 to Fodor et al. High density nucleic acid arrays can also befabricated by depositing premade or natural nucleic acids inpredetermined positions. Synthesized or natural nucleic acids aredeposited on specific locations of a substrate by light directedtargeting and oligonucleotide directed targeting. Another embodimentuses a dispenser that moves from region to region to deposit nucleicacids in specific spots.

Oligonucleotide probe arrays for expression monitoring can be made andused according to any techniques known in the art (see for example,Lockhart et al., Nat. Biotechnol. 14, 1675-1680 (1996); McGall et al.,Proc. Nat. Acad. Sci. USA 93, 13555-13460 (1996). Such probe arrays maycontain at least two or more oligonucleotides that are complementary toor hybridize to two or more of the genes described herein. Such arraysmay also contain oligonucleotides that are complementary to or hybridizeto at least 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 70 or more of the genesdescribed therein.

Gene Signature Differential Analysis.

Gene Signature Differential analysis is a method designed to detectgenes present in one sample set, and absent in another. Genes withdifferential expression in cells from sites of infection or inflammationversus normal tissue are better diagnostic and therapeutic targets thangenes that do not change in expression.

Hybridization

Nucleic acid hybridization simply involves contacting a probe and targetnucleic acid under conditions where the probe and its complementarytarget can form stable hybrid duplexes through complementary basepairing (see WO 99/32660 to Lockhart). The nucleic acids that do notform hybrid duplexes are then washed away leaving the hybridized nucleicacids to be detected, typically through detection of an attacheddetectable label. It is generally recognized that nucleic acids aredenatured by increasing the temperature or decreasing the saltconcentration of the buffer containing the nucleic acids. Under lowstringency conditions (e.g., low temperature and/or high salt) hybridduplexes (e.g., DNA-DNA, RNA-RNA or RNA-DNA) will form even where theannealed sequences are not perfectly complementary. Thus, specificity ofhybridization is reduced at lower stringency. Conversely, at higherstringency (e.g., higher temperature or lower salt) successfulhybridization requires fewer mismatches. One of skill in the art willappreciate that hybridization conditions may be selected to provide anydegree of stringency. In a preferred embodiment, hybridization isperformed at low stringency, in this case in 6×SSPE-T at 37° C., (0.005%Triton x-100) to ensure hybridization and then subsequent washes areperformed at higher stringency (e.g., 1×SSPE-T at 37° C.) to eliminatemismatched hybrid duplexes. Successive washes may be performed atincreasingly higher stringency (e.g. down to as low as 0.25×SSPE-T at37° C. to 50° C. until a desired level of hybridization specificity isobtained. Stringency can also be increased by addition of agents such asformamide. Hybridization specificity may be evaluated by comparison ofhybridization to the test probes with hybridization to the variouscontrols that can be present (e.g., expression level controls,normalization controls, mismatch controls, etc.).

In general, there is a tradeoff between hybridization specificity(stringency) and signal intensity. Thus, in a preferred embodiment, thewash is performed at the highest stringency that produces consistentresults and that provides a signal intensity greater than approximately10% of the background intensity. The hybridized array may be washed atsuccessively higher stringency solutions and read between each wash.Analysis of the data sets thus produced will reveal a wash stringencyabove which the hybridization pattern is not appreciably altered andwhich provides adequate signal for the particular oligonucleotide probesof interest.

Signal Detection

The hybridized nucleic acids are typically detected by detecting one ormore labels attached to the sample nucleic acids. The labels may beincorporated by any of a number of means well known to those of skill inthe art (see WO 99/32660 to Lockhart).

Any suitable methods can be used to detect one or more of the markersdescribed herein. For example, gas phase ion spectrometry can be used.This technique includes, e.g., laser desorption/ionization massspectrometry. In some embodiments, the sample can be prepared prior togas phase ion spectrometry, e.g., pre-fractionation, two-dimensional gelchromatography, high performance liquid chromatography, etc. to assistdetection of markers. Detection of markers can be achieved using methodsother than gas phase ion spectrometry. For example, traditionalimmunoassays (e.g., ELISA) can be used to detect the markers in asample. These detection methods are described in detail below.

Detection by Gas Phase Ion Spectrometry

Markers present in a sample can also be detected using gas phase ionspectrometry, and more preferably, using mass spectrometry. In oneembodiment, matrix-assisted laser desorption/ionization (“MALDI”) massspectrometry can be used. In MALDI, the sample is typicallyquasi-purified to obtain a fraction that essentially consists of amarker or markers using protein separation methods such astwo-dimensional gel electrophoresis or high performance liquidchromatography (HPLC).

In another embodiment, surface-enhanced laser desorption/ionization massspectrometry (“SELDI”) can be used. SELDI uses a substrate comprisingadsorbents to capture markers, which can then be directly desorbed andionized from the substrate surface during mass spectrometry. Since thesubstrate surface in SELDI captures markers, a sample need not bequasi-purified as in MALDI. However, depending on the complexity of asample and the type of adsorbents used, it may be desirable to prepare asample to reduce its complexity prior to SELDI analysis.

Various sample preparation methods to assist detection of markers in asample and gas phase ion spectrometry methods are described in detailbelow. Optionally, one or combination of methods described below orother methods known in the art can be used to prepare a sample tofurther assist detection and characterization of markers in a sample. Insome embodiments, a sample can be pre-fractionated to provide a lesscomplex sample prior to gas phase ion spectrometry analysis. Moreover,pre-fractionation protocols can provide additional information regardingphysical and chemical characteristics of markers. For example, if asample was pre-fractionated using an anion-exchange spin column, and ifa marker is eluted at a certain pH, this elution characteristic providesinformation regarding binding properties of the marker. In anotherexample, a sample can be pre-fractionated by removing proteins or othermolecules in the sample that are present in a high quantity or that mayinterfere with the detection of markers in a sample. In anotherembodiment, a sample can be pre-fractionated according to size ofproteins in a sample using size exclusion chromatography. For abiological sample wherein the amount of sample available is small,preferably a size selection spin column is used. For example, K-30 spincolumn (Ciphergen Biosystems, Inc.) can be used. In general, the firstfraction that is eluted from the column (“fraction 1”) has the highestpercentage of high molecular weight proteins; fraction 2 has a lowerpercentage of high molecular weight proteins; fraction 3 has even alower percentage of high molecular weight proteins; fraction 4 has thelowest amount of large proteins; and so on. Each fraction can then beanalyzed by gas phase ion spectrometry for the detection of markers. Instill another embodiment, biomolecules in a sample can be separated byhigh-resolution electrophoresis, e.g., one or two-dimensional gelelectrophoresis. A fraction containing a marker can be isolated andfurther analyzed by gas phase ion spectrometry. Preferably,two-dimensional gel electrophoresis is used to generate two-dimensionalarray of spots of biomolecules, including one or more markers. See,e.g., Jungblut and Thiede, Mass Spectr. Rev. 16:145-162 (1997).

The two-dimensional gel electrophoresis can be performed using methodsknown in the art. See, e.g., Deutseher ed., Methods In Enzymology vol.182. Typically, biomolecules in a sample are separated by, e.g.,isoelectric focusing, during which biomolecules in a sample areseparated in a pH gradient until they reach a spot where their netcharge is zero (i.e., isoelectric point). This first separation stepresults in one-dimensional array of biomolecules. The biomolecules inone dimensional array is further separated using a technique generallydistinct from that used in the first separation step. For example, inthe second dimension, biomolecules separated by isoelectric focusing arefurther separated using a polyacrylamide gel, such as polyacrylamide gelelectrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE),SDS-PAGE gel allows further separation based on molecular mass ofbiomolecules. Typically, two-dimensional gel electrophoresis canseparate chemically different biomolecules in the molecular mass rangefrom 1000-200,000 Da within complex mixtures.

Biomolecules in the two-dimensional array can be detected using anysuitable methods known in the art. For example, biomolecules in a gelcan be labeled or stained (e.g. Coomassie Blue or silver staining). Ifgel electrophoresis generates spots that correspond to the molecularweight of one or more markers of the invention, the spot can be isfurther analyzed by gas phase ion spectrometry. For example, spots canbe excised from the gel and analyzed by gas phase ion spectrometry.Alternatively, the gel containing biomolecules can be transferred to aninert membrane by applying an electric field. Then a spot on themembrane that approximately corresponds to the molecular weight of amarker can be analyzed by gas phase ion spectrometry. In gas phase ionspectrometry, the spots can be analyzed using any suitable techniques,such as MALDI or SELDI (e.g., using ProteinChip® array) as described indetail below.

Prior to gas phase ion spectrometry analysis, it may be desirable tocleave biomolecules in the spot into smaller fragments using cleavingreagents, such as proteases (e.g., trypin). The digestion ofbiomolecules into small fragments provides a mass fingerprint of thebiomolecules in the spot, which can be used to determine the identity ofmarkers if desired.

High Performance Liquid Chromatography

In yet another embodiment, high performance liquid chromatography (HPLC)can be used to separate a mixture of biomolecules in a sample based ontheir different physical properties, such as polarity, charge and size.HPLC instruments typically consist of a reservoir of mobile phase, apump, an injector, a separation column, and a detector. Biomolecules ina sample are separated by injecting an aliquot of the sample onto thecolumn. Different biomolecules in the mixture pass through the column atdifferent rates due to differences in their partitioning behaviorbetween the mobile liquid phase and the stationary phase. A fractionthat corresponds to the molecular weight and/or physical properties ofone or more markers can be collected. The fraction can then be analyzedby gas phase ion spectrometry to detect markers. For example, the spotscan be analyzed using either MALDI or SELDI (e.g., using ProteinChip®array).

Desorption/Ionization and Detection

Markers can be ionized using gas phase ion spectrometry. Any suitablegas phase ion spectrometers can be used as long as it allows markers onthe substrate to be resolved. Preferably, gas phase ion spectrometersallow quantitation of markers. In one embodiment, a gas phase ionspectrometer is a mass spectrometer. In a typical mass spectrometer, asubstrate or a probe comprising markers on its surface is introducedinto an inlet system of the mass spectrometer. The markers are thendesorbed by a desorption source such as a laser, fast atom bombardment,high energy plasma, electrospray ionization, thermospray ionization,liquid secondary ion MS, field desorption, etc. The generated desorbed,volatilized species consist of preformed ions or neutrals which areionized as a direct consequence of the desorption event. Generated ionsare collected by an ion optic assembly, and then a mass analyzerdisperses and analyzes the passing ions. The ions editing the massanalyzer are detected by a detector. The detector then translatesinformation of the detected ions into mass-to-charge ratios. Detectionof the presence of markers or other substances will typically involvedetection of signal intensity. This, in turn, can reflect the quantityand character of markers bound to the substrate. In laser desorptionmass spectrometry, a substrate or a probe comprising markers isintroduced into an inlet system. The markers are desorbed and ionizedinto the gas phase by laser from the ionization source. The ionsgenerated are collected by an ion optic assembly, and then in atime-of-flight mass analyzer, ions are accelerated through a short highvoltage field and let drift into a high vacuum chamber. At the far endof the high vacuum chamber, the accelerated ions strike a sensitivedetector surface at a different time. Since the time-of-flight is afunction of the mass of the ions, the elapsed time between ion formationand ion detector impact can be used to identify the presence or absenceof markers of specific mass to charge ratio. In another embodiment, anion mobility spectrometer can be used to detect markers. The principleof ion mobility spectrometry is based on different mobility of ions.Specifically, ions of a sample produced by ionization move at differentrates, due to their difference in, e.g., mass, charge, or shape, througha tube under the influence of an electric field. The ions (typically inthe form of a current) are registered at the detector which can then beused to identify a marker or other substances in a sample. One advantageof ion mobility spectrometry is that it can operate at atmosphericpressure. In yet another embodiment, a total ion current measuringdevice can be used to detect and characterize markers. This device canbe used when the substrate has only a single type of marker. When asingle type of marker is on the substrate, the total current generatedfrom the ionized marker reflects the quantity and other characteristicsof the marker. The total ion current produced by the marker can then becompared to a control (e.g., a total ion current of a known compound).The quantity or other characteristics of the marker can then bedetermined.

Detection by Immunoassay

In another embodiment, an immunoassay can be used to detect and analyzemarkers in a sample. This method comprises: (a) providing an antibodythat specifically binds to a marker; (b) contacting a sample with theantibody; and (c) detecting the presence of a complex of the antibodybound to the marker in the sample. To prepare an antibody thatspecifically binds to a marker, purified markers or their nucleic acidsequences can be used. Nucleic acid and amino acid sequences for markerscan be obtained by further characterization of these markers. Using thepurified markers or their nucleic acid sequences, antibodies thatspecifically bind to a marker can be prepared using any suitable methodsknown in the art. See, e.g., Cagan, Current Protocols in Immunology(1991); Harlow & Lane, Antibodies: A Laboratory Manual (1988); Goding,Monoclonal Antibodies: Principles and Practice (2d ed. 1986); and Kohler& Milstein, Nature 256:495-497 (1975). Such techniques include, but arenot limited to, antibody preparation by selection of antibodies fromlibraries of recombinant antibodies in phage or similar vectors, as wellas preparation of polyclonal and monoclonal antibodies by immunizingrabbits or mice (see, e.g., Huse et al., Science 246:1275-1281 (1989);Ward et al., Nature 341:544-546 (1989)).

After the antibody is provided, a marker can be detected and/orquantified using any of suitable immunological binding assays known inthe art (see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and4,837,168). Useful assays include, for example, an enzyme immune assay(EIA) such as enzyme-linked immunosorbent assay (ELISA), a radioimmuneassay (RIA), a Western blot assay, or a slot blot assay. These methodsare also described in, e.g., Methods in Cell Biology: Antibodies in CellBiology, volume 37 (Asai, ed. 1993); Basic and Clinical Immunology(Stites & Terr, eds., 7th ed. 1991); and Harlow & Lane, supra.

Generally, a sample obtained from a subject can be contacted with theantibody that specifically binds the marker. Optionally, the antibodycan be fixed to a solid support to facilitate washing and subsequentisolation of the complex, prior to contacting the antibody with asample. Examples of solid supports include glass or plastic in the formof, e.g., a microtiter plate, a stick, a bead, or a microbead.Antibodies can also be attached to a probe substrate or ProteinChip®array and can be analyzed by gas phase ion spectrometry as describedabove. The sample is preferably a biological fluid sample taken from asubject. Examples of biological samples include urine, barbotage, blood,serum, plasma, tears, saliva, cerebrospinal fluid, urine, tissue, etc.In a preferred embodiment, the biological fluid comprises synovialfluid. The sample can be diluted with a suitable diluent beforecontacting the sample to the antibody.

After incubating the sample with antibodies, the mixture is washed andthe antibody-marker complex formed can be detected. This can beaccomplished by incubating the washed mixture with a detection reagent.This detection reagent may be, e.g., a second antibody which is labeledwith a detectable label. Exemplary detectable labels include magneticbeads (e.g., DYNABEADS™), fluorescent dyes, radiolabels, enzymes (e.g.,horse radish peroxide, alkaline phosphatase and others commonly used inan ELISA), and colorimetric labels such as colloidal gold or coloredglass or plastic beads. Alternatively, the marker in the sample can bedetected using an indirect assay, wherein, for example, a second,labeled antibody is used to detect bound marker-specific antibody,and/or in a competition or inhibition assay wherein, for example, amonoclonal antibody which binds to a distinct epitope of the marker isincubated simultaneously with the mixture.

Databases

Databases may contain information associated with a given cell or tissuesample such as descriptive information concerning the clinical status ofthe tissue sample, or the patient from which the sample was derived. Thedatabase may be designed to include different parts, for instance asequences database and a gene expression database. Methods for theconfiguration and construction of such databases are widely available,for instance, see U.S. Pat. No. 5,953,727 Akerblom et al. Anyappropriate computer platform may be used to perform the necessarycomparisons between sequence information, gene expression informationand any other information in the database or provided as an input. Forexample, a large number of computer workstations are available from avariety of manufacturers, such has those available from SiliconGraphics. Client-server environments, database servers and networks arealso widely available and appropriate platforms for the databases of theinvention. The databases may be used to produce, among other things,electronic Northerns to allow the user to determine the cell type ortissue in which a given gene is expressed and to allow determination ofthe abundance or expression level of a given gene in a particular tissueor cell.

Patterns can be compared manually (by a person) or by a computer orother machine. An algorithm can be used to detect similarities anddifferences. The algorithm may score and compare, for example, the geneswhich are expressed and the genes which are not expressed.Alternatively, the algorithm may look for changes in intensity ofexpression of a particular gene and score changes in intensity betweentwo samples. A variety of such algorithms are known in the art.Similarities may be determined on the basis of genes which are expressedin both samples and genes which are not expressed in both samples or onthe basis of genes whose intensity of expression are numericallysimilar. Differences are considered significant when they are greaterthan 2-fold, 3-fold or 5-fold from the base value. Alternatively, amathematical approach can be used to conclude whether differences in thegene expression exhibited by different samples is significant (see,e.g., Golub et al., Science 286, 531 (1999). One approach to determinewhether a sample is more similar to or has maximum similarity with agiven condition (e.g., a particular grade or stage of tumor progression)is to compare the Euclidean distances (see Golub et al. and Example 6)between the sample and one or more pools representing differentconditions for comparison; the pool with the smallest vector angle isthen chosen as the most similar to the test sample among the poolscompared.

Diagnostic Kits

Kits can be prepared for use within any of the above diagnostic methods.Such kits typically include two or more components necessary forperforming a diagnostic assay Components may be compounds, reagents,containers and/or equipment. For example, one container within a kit maycontain a monoclonal antibody or fragment thereof that specificallybinds to protein found to be differentially expressed in a specific typeof local inflammation. Such antibodies or fragments may be providedattached to a support material, as described above. One or moreadditional containers may enclose elements, such as reagents or buffers,to be used in the assay. Such kits may also, or alternatively, contain adetection reagent as described above that contains a reporter groupsuitable for direct or indirect detection of antibody binding.

Alternatively, a kit may be designed to detect the level of mRNAencoding a protein in a biological sample. Such kits generally compriseat least one oligonucleotide probe or primer, as described above, thathybridizes to a polynucleotide encoding a lung tumor protein. Such anoligonucleotide may be used, for example, within a PCR or hybridizationassay. Additional components that may be present within such kitsinclude a second oligonucleotide and/or a diagnostic reagent orcontainer to facilitate the detection of a polynucleotide encoding aprotein.

In one example, a patient has an acutely inflamed total kneearthroplasty site. The knee effusion is aspirated and RNA is isolatedand purified from the fluid's cellular content. The gene expression ofcells in the fluid is determined using an Affymetrix® U133A Humanmicroarray chip. The pattern of gene expression (all or one or more ofthose identified as described in the examples by comparison to knowncontrols) is found to match the pattern exhibited by other cases ofacute infection. Specific microarrays could be generated and included ina kit, allowing the specific evaluation of genes known to bedifferentially expressed in local inflammatory conditions. In anotherexample, a patient has an acutely inflamed total knee arthroplasty site.The knee effusion is aspirated and the fluid is stored in a fashion thatpreserves overall protein integrity. The inflamed fluid is used as thesample for ELISA testing. The sample can be diluted before testing.ELISA testing is directed at detection of proteins that are products ofgenes found to be differentially expressed in various forms of localinflammation. The protein expression levels, measured by ELISA, arecompared to sample standards in order to assign a diagnosis. A kit couldbe provided, utilizing immunochemistry in fluid or solid state, tomeasure the levels of a specific set of genes to aid in the diagnosis oflocal inflammatory diseases. In yet another example, a sample isattained through biopsy or surgical excision. It may be prepared in avariety of methods including frozen section, paraffin embedding, etc.Once on a slide, it is stained with antibody and marker preparations. Anantibody to a gene that is upregulated in a specific form ofinflammation is used to stain the sample. The white blood cells (“WBC”)on the slide are then analyzed for evidence of gene expression based onprotein binding. A single or combination of antibodies could be used onsolid tissue, in an effort to diagnose the underlying etiology ofinflammation. They could be provided together in a kit for thepathologic diagnosis of local acute inflammatory diseases andconditions.

The following examples are offered by way of illustration and not by wayof limitation.

Example 1 Method of RNA isolation from Synovial Fluid Lymphocytes

Suitable RNA stabilizing solutions are commercially available, forexample, from Ambion, Inc.

-   -   1. Synovial fluid is added to RNAlater® (Ambion Inc.) at a 1:3        ratio and is immediately mixed.    -   2. The mixture is stable at room temperature for 3 hours, in a        clinical refrigerator for 1 day and in a freezer indefinitely.    -   3. Centrifuge 2 cc sample for 1 minute in a microfuge,    -   4. Remove the supernatant and add 800 μl lysis fluid (Ambion        Inc.), and 50 μl of sodium acetate solution (Ambion Inc.)    -   5. Resuspend pellet with an 18 g needle and syringe.    -   6. Add 500 μl of Acid-phenol:chloroform and vortex 30 seconds.    -   7. Incubate at room temperature for 5 min, then microfuge for 10        min.    -   8. Transfer upper aqueous phase to a new tube.    -   9. Add ½ volume of 100% ethanol and mix.    -   10. Pass 9 through a filter cartridge (Ambion Inc.) and wash        with 700 μl of wash solution 1 ((Ambion Inc.).    -   11. Wash 2× with 700 μl of wash solution ⅔ (Ambion Inc.)    -   12. Spin on microfuge for 1 minute to dry.    -   13. Elute with elution buffer (Ambion Inc.)

Example 2 Isolating and Evaluating RNA from Inflammatory Fluids, for thePurposes of Identifying Differential Expression

This example describes the identification of genes that aredifferentially expressed in the acute synovial fluid infiltrate thatresults after S. aureus joint infection. The acute synovial infiltrateresulting from gout is used as an aseptic control.

Synovial fluid samples were aspirated from seven patients with acute S.aureus knee infections and five patients with acute gout of the knee.Some patients had total knee arthroplasties. AU patients in both groupshad approximately 90% neutrophils in the synovial fluid. All patientswith S. aureus infection exhibited fever, acute arthritis, multiplepositive S. aureus cultures, high CRP values (average, 22; range,1.9-34), high ESR (average, 90; range, 39-125), and an elevated synovialWBC count (average, 90,000 cells/mm³; range, 27,000-183,000; averagedifferential, 93% neutrophils). Five samples were from infected totalknee arthroplasties and two samples were from infected native knees. AUpatient samples with gout revealed monosodium urate crystals, elevatedsynovial WBC count (average, 10,000; range, 800-16,000, averagedifferential 90% neutrophils) and clinical resolution withoutantibiotics. All samples were in native knees.

The synovial fluid was added in a 1:3 ratio to RNAlater (Ambion Inc.)for stabilization and preservation of the RNA profile in theinflammatory cells of the fluid, as described in example 1. The mixturewas agitated and stored at 20 degrees Celsius for eventual isolation andpurification of RNA. At a later date, each sample mixture wascentrifuged for collection of the inflammatory cells. The pellet wasresuspended in a homogenization buffer and agitated by needle techniqueusing a 10 cc syringe and an 18 gauge needle. Phenol:chloroformextraction techniques were used to create an aqueous layer of fluidcontaining partially purified RNA. The aqueous layer was then applied toan RNA binding filter (Ambion Inc.) attached to a microfuge tube. Thebound RNA was washed with a variety of detergent buffers and finallyeluted with hot elution buffer. The resulting fluid was assessed byspectrophotometry and the Agilent bioanalyzer for RNA purity andquantity. The RNA was found to be pure.

The RNA samples were then applied to Affymetrix® microarrays (U133A).All protocols were conducted as described in the Affymetrix® GeneChipExpression Analysis Technical Manual. Briefly, 5 μg of total RNA wasconverted to first-strand cDNA using Superscript II reversetranscriptase. Second-strand cDNA synthesis was followed by in vitrotranscription for linear amplification of each transcript andincorporation of biotinylated CTP and UTP. The cRNA products werefragmented to 200 nucleotides or less, and hybridized for 16 hours tothe microarrays. The microarrays were then washed at low (6×SSPE) andhigh (100 mM MES, 0.1M NaCl) stringency and stained withstreptavidin-phycoerythrin. Fluorescence was amplified by addingbiotinylated anti-streptavidin and an additional aliquot ofstreptavidin-phycoerythrin stain. A confocal scanner was used to collectfluorescence signal at 3 um resolution after excitation at 570 nm. Theaverage signal from two sequential scans was calculated for eachmicroarray feature. It was found that even though the two groups (goutand S. aureus) had similar cell and differential counts on synovialfluid analysis, the gene expression signatures in these cells was verydifferent. Tables I and II list the gene expression in order of greatestchange. About 1600 genes showed a statistically different expressionlevel between the two groups, as shown in Table I (upregulation inseptic versus aseptic inflammation) and Table II (downregulation inseptic versus aseptic inflammation). As indicated in Tables I and II,“source” can be inputted into the Internet (GeneBank) or throughAffymetrix® to obtain the name, description and other identifyinginformation relating to each gene and encoded product. The results areordered by degree of change in expression.

This study established that one can analyze white blood cell orneutrophil gene expression as a diagnostic tool anywhere in the body orusing a sample extracted from the body, for example, in synovial fluid.More specifically, it has been demonstrated in vivo, that an acute whiteblood cell infiltrate is associated with a gene expression profile thatis specific for the underlying source of inflammation.

Example 3 Identification of Genes that are Differentially ExpressedBetween Patients with Acute Infection and Aseptic Loosening

The materials and methods are identical to those described in Example 1with the following exceptions.

Genes were identified that are differentially expressed in acutesynovial fluid infiltrate between patients with acute infection andpatients with aseptic loosening of total knee arthroplasty. Asepticloosening is a loosening of the total joint without involvement ofbacteria. Five patients with acute infection, which met strict inclusioncriteria including purulence, high synovial white blood cell (WBC)count, high Erythrocyte sedimentation rate (ESR)/C-reactive protein(CRP) lab values, and positive microbial culture were analyzed. Elevenpatients with aseptic loosening, which had low ESR/CRP lab values, lowsynovial WBC count, negative microbial cultures, and x-ray evidence of aloose arthroplasty were analyzed.

RNA was isolated from the cells in the synovial aspirate, and preparedfor analysis with the Affymetrix® U133 Plus 2.0 array as describedabove. The data was analyzed by similar methods as described in Example2 to identify differentially expressed genes.

As expected, many genes were differentially expressed between patientswith acute infection and patients with aseptic loosening. 2451 genesshowed a statistically different level of expression between the twogroups when setting the false discovery rate to 5% and using rankproducts analysis (All raw data was sent to the to The Sir Henry WelcomeFunctional Genomics Facility & Bioinformatics Research Centre at GlasgowUniversity).

Many of the genes identified in Example 1, were also identified as beingdifferentially expressed between patients with acute infection andpatients with aseptic loosening as described in this example. Theseresults demonstrate that global markers specific for the underlyingsource of infection are produced by WBCs and have diagnostic value. Allof the previously identified genes have potential diagnostic value inidentifying clinical cases of infection. Genes that were differentiallyexpressed in Example 1 and in the present example include, but are notlimited to, skin-derived antileukoproteinase (SKALP) (PI3),interleukin-1beta (IL1B), interleukin-8 (IL8), Interleukin-1receptor-associated kinase 3 (IRAK3), CC chemokine ligand 3 (CCL3), CCchemokine ligand 4 (CCL4), superoxide dismutase 2 (SOD2), Nuclear Factorof Kappa light polypeptide gene enhancer in B-cells Inhibitor, Alpha(NFKBIA), Nijmegen breakage syndrome 1 (NBS1), tumor necrosis factoralpha-induced protein 6 (TNFAIP6), and plasminogen activator, urokinase(PLAU).

As expected from the results in Example 1, these results demonstratethat genes expressed from WBCs at the site of local inflammation havediagnostic value in terms of identifying the etiology of localinflammation. In other words, these results demonstrate that determiningthe level of expression of one or more genes in a clinical sampleobtained from a site of local inflammation facilitates diagnosis of thesource of that inflammation.

TABLE I Genes whose Expression is Upregulated in Septic as compared toAseptic Inflammation Affymetrix ® Fold Gene Rank U133A name Change“Source” symbol 1 203691_at −19.55 NM_002638 PI3 2 206025_s_at −13.37AW188198 TNFAIP6 3 221345_at −8.96 NM_005306 GPR43 4 202269_x_at −9.81BC002666 GBP1 5 204103_at −13.31 NM_002984 CCL4 6 205114_s_at −9.83NM_002983 CCL3 7 205220_at −6.94 NM_006018 HM74 8 41469_at −10.79 L10343PI3 9 36711_at −9.5 AL021977 MAFF 10 205479_s_at −9.94 NM_002658 PLAU 11204224_s_at −10.78 NM_000161 GCH1 12 215078_at −9.41 AL050388 SOD2 13203021_at −10.19 NM_003064 SLPI 14 218507_at −8.61 NM_013332 HIG2 15212657_s_at −8.38 U65590 IL1RN 16 202643_s_at −6.63 AI738896 TNFAIP3 17209969_s_at −6.06 BC002704 STAT1 18 206067_at −5.89 NM_000576 IL1B 19202193_at −6.66 NM_005569 LIMK2 20 202270_at −7.5 NM_002053 GBP1 21202905_x_at −6.62 AI796269 NBS1 22 39402_at −5.36 M15330 IL1B 23206026_s_at −6.08 NM_007115 TNFAIP6 24 202906_s_at −7.46 AF049895 NBS125 211668_s_at −7.87 K03226 PLAU 26 201502_s_at −6.66 AI078167 NFKBIA 27217299_s_at −6.18 AK001017 NBS1 28 205040_at −8.1 NM_000607 ORM1 29205041_s_at −8.1 NM_000607 ORM1 30 203470_s_at −4.99 AI433595 PLEK 31216841_s_at −4.44 X15132 SOD2 32 215223_s_at −4.64 W46388 SOD2 33211434_s_at −6.39 AF015524 CCRL2 34 202887_s_at −4.3 NM_019058 RTP801 35214974_x_at −4.5 AK026546 CXCL5 36 202644_s_at −5.36 NM_006290 TNFAIP337 218280_x_at −5.2 NM_003516 — 38 207196_s_at −5.4 NM_006058 TNIP1 39209774_x_at −6.47 M57731 CXCL2 40 213524_s_at −4.47 NM_015714 G0S2 41202638_s_at −4.29 NM_000201 ICAM1 42 209288_s_at −4.57 AL136842 CDC42EP343 205863_at −4.37 NM_005621 S100A12 44 212090_at −4.71 AL571424 GRINA45 205013_s_at −6.03 NM_000675 ADORA2A 46 218810_at −4.96 NM_025079FLJ23231 47 209050_s_at −5.93 AI421559 RALGDS 48 202581_at −8.84NM_005346 HSPA1A 49 214290_s_at −4 AI313324 — 50 210582_s_at −3.78AL117466 LIMK2 51 207907_at −5.1 NM_003807 TNFSF14 52 209051_s_at −5.06AF295773 RALGDS 53 205476_at −5.25 NM_004591 CCL20 54 201169_s_at −4.62BG326045 BHLHB2 55 201489_at −5.1 BC005020 PPIF 56 221920_s_at −3.44BE677761 MSCP 57 206637_at −4.81 NM_014879 GPR105 58 204351_at −4.19NM_005980 S100P 59 207574_s_at −3.81 NM_015675 GADD45B 60 210845_s_at−4.65 U08839 PLAUR 61 213716_s_at −4.18 BF939675 SECTM1 62 222088_s_at−2.67 AA778684 SLC2A14 63 209038_s_at −4.31 AL579035 EHD1 64 213638_at−3.75 AW054711 RPEL1 65 212659_s_at −3.49 AW083357 IL1RN 66 218880_at−4.58 N36408 FOSL2 67 222221_x_at −3.74 AY007161 EHD1 68 202014_at −4.22NM_014330 PPP1R15A 69 202531_at −4.54 NM_002198 IRF1 70 207850_at −4.67NM_002090 CXCL3 71 209305_s_at −3.94 AF078077 GADD45B 72 202748_at −3.6NM_004120 GBP2 73 204908_s_at −3.83 NM_005178 BCL3 74 200800_s_at −4.78NM_005345 HSPA1A 75 204279_at −3.68 NM_002800 PSMB9 76 209304_x_at −3.43AF087853 GADD45B 77 202637_s_at −3.5 AI608725 — 78 218695_at −4.77NM_019037 RRP41 79 202284_s_at −4.22 NM_000389 CDKN1A 80 37028_at −4.18U83981 PPP1R15A 81 220034_at −3.63 NM_007199 IRAK3 82 209369_at −4.09M63310 ANXA3 83 208112_x_at −3.48 NM_006795 EHD1 84 202307_s_at −3.7NM_000593 TAP1 85 204698_at −3.33 NM_002201 ISG20 86 216243_s_at −3.02BE563442 IL1RN 87 218999_at −3.73 NM_018295 FLJ11000 88 201490_s_at−3.14 NM_005729 PPIF 89 206420_at −3.33 NM_005849 IGSF6 90 205681_at−2.92 NM_004049 BCL2A1 91 202912_at −3.19 NM_001124 ADM 92 202499_s_at−2.34 NM_006931 SLC2A3 93 209039_x_at −3.3 AF001434 EHD1 94 211924_s_at−3.37 AY029180 PLAUR 95 211883_x_at −3.1 M76742 CEACAM1 96 217475_s_at−2.73 AC002073 LIMK2 97 211725_s_at −2.85 BC005884 BID 98 216782_at−2.75 AK026679 — 99 209498_at −3.12 X16354 CEACAM1 100 209037_s_at −3.13AW182860 EHD1 101 201810_s_at −3.21 AL562152 SH3BP5 102 202426_s_at−3.44 BE675800 RXRA 103 58696_at −3.85 AL039469 RRP41 104 33304_at −3.05U88964 ISG20 105 203045_at −3.22 NM_004148 NINJ1 106 214657_s_at −3.2AU134977 — 107 205270_s_at −3.16 NM_005565 LCP2 108 214414_x_at −3.19T50399 HBA1 109 203137_at −3.15 NM_004906 WTAP 110 219938_s_at −3.85NM_024430 PSTPIP2 111 219669_at −3.39 NM_020406 PRV1 112 211372_s_at−2.67 U64094 IL1R2 113 200986_at −4.04 NM_000062 SERPING1 114202907_s_at −3.23 NM_002485 NBS1 115 201649_at −3.45 NM_004223 UBE2L6116 201601_x_at −2.62 NM_003641 IFITM1 117 203234_at −3.05 NM_003364UPP1 118 222303_at −2.74 AV700891 ETS2 119 213812_s_at −3.13 AK024748CAMKK2 120 218033_s_at −2.78 NM_003498 SNN 121 204470_at −2.93 NM_001511CXCL1 122 217497_at −2.97 AW613387 ECGF1 123 216236_s_at −2.97 AL110298SLC2A14 124 204070_at −3.17 NM_004585 RARRES3 125 201473_at −2.78NM_002229 JUNB 126 205269_at −2.9 AI123251 LCP2 127 205322_s_at −2.81AW182367 — 128 204951_at −2.36 NM_004310 ARHH 129 202498_s_at −2.63BE550486 SLC2A3 130 205403_at −2.31 NM_004633 IL1R2 131 203471_s_at−3.01 NM_002664 PLEK 132 215977_x_at −3.16 X68285 GK 133 215966_x_at−3.11 AA292874 GK 134 211527_x_at −3.15 M27281 VEGF 135 217977_at −2.57NM_016332 SEPX1 136 204780_s_at −2.28 AA164751 TNFRSF6 137 213700_s_at−3.04 AA554945 PKM2 138 206765_at −3.02 AF153820 KCNJ2 139 203936_s_at−3.19 NM_004994 MMP9 140 207111_at −2.42 NM_001974 EMR1 141 204748_at−2.54 NM_000963 PTGS2 142 41386_i_at −3.01 AB002344 KIAA0346 143201294_s_at −2.71 N24643 WSB1 144 200887_s_at −3.36 NM_007315 STAT1 145209367_at −3.08 AB002559 STXBP2 146 91684_g_at −2.81 AI571298 RRP41 147217167_x_at −3.08 AJ252550 — 148 202497_x_at −2.91 AI631159 SLC2A3 149208829_at −2.35 AF029750 TAPBP 150 209286_at −2.52 AI754416 CDC42EP3 151215760_s_at −2.85 AC005390 KIAA0963 152 202807_s_at −2.83 NM_005488 TOM1153 205781_at −2.6 NM_004913 C16orf7 154 214022_s_at −2.27 AA749101IFITM1 155 219622_at −2.52 NM_017817 RAB20 156 204961_s_at −2.89NM_000265 NCF1 157 201362_at −2.85 AF205218 IVNS1ABP 158 210119_at −2.31U73191 KCNJ15 159 220712_at −2.51 NM_024984 — 160 210285_x_at −2.4BC000383 WTAP 161 202464_s_at −2.62 NM_004566 PFKFB3 162 217995_at −2.71NM_021199 SQRDL 163 210512_s_at −3.03 AF022375 VEGF 164 209803_s_at −4.1AF001294 PHLDA2 165 209310_s_at −2.76 U25804 CASP4 166 208092_s_at −2.21NM_030797 DKFZP566A1524 167 208992_s_at −2.42 BC000627 STAT3 168200666_s_at −3.28 NM_006145 DNAJB1 169 200999_s_at −2.76 NM_006825 CKAP4170 213038_at −2.29 AL031602 FLJ90005 171 214637_at −1.98 BG437034 OSM172 208749_x_at −2.35 AF085357 FLOT1 173 220193_at −2.69 NM_024676FLJ22938 174 211965_at −2.33 BE620915 ZFP36L1 175 205205_at −2.5NM_006509 RELB 176 204116_at −2.62 NM_000206 IL2RG 177 201811_x_at −2.5NM_004844 SH3BP5 178 207535_s_at −2.5 NM_002502 NFKB2 179 218618_s_at−2.93 NM_022763 FAD104 180 203068_at −2.47 NM_014851 KIAA0469 181201668_x_at −2.35 AW163148 MARCKS 182 209282_at −2.68 AF309082 PRKD2 183215498_s_at −2.83 AA780381 MAP2K3 184 211745_x_at −2.39 BC005931 HBA1185 202121_s_at −2.32 NM_014453 BC-2 186 204669_s_at −2.6 NM_007219RNF24 187 221477_s_at −2.59 BF575213 SOD2 188 220000_at −2.48 NM_003830SIGLEC5 189 201631_s_at −2.3 NM_003897 IER3 190 210142_x_at −2.35AF117234 FLOT1 191 213146_at −2.48 AA521267 KIAA0346 192 209636_at −2.68BC002844 NFKB2 193 211316_x_at −2.61 AF009616 CFLAR 194 201772_at −2.02NM_015878 OAZIN 195 212492_s_at −2.47 AW237172 KIAA0876 196 214722_at−2.88 AW516297 NOTCH2 197 203897_at −2.33 BE963444 LOC57149 198202102_s_at −2.7 BF718610 BRD4 199 204494_s_at −2.65 AW516789DKFZP434H132 200 210563_x_at −2.25 U97075 CFLAR 201 205443_at −2.26NM_003082 SNAPC1 202 AFFX- −2.09 AFFX- — HUMISGF3A/ HUMISGF3A/M97935_MA_at M97935_MA 203 AFFX- −2.79 AFFX- — HUMISGF3A/ HUMISGF3A/M97935_3_at M97935_3 204 221903_s_at −2.51 BE046443 CYLD 205 209458_x_at−2.29 AF105974 HBA1 206 212723_at −2.45 AK021780 PTDSR 207 202510_s_at−2.7 NM_006291 TNFAIP2 208 210511_s_at −2.73 M13436 INHBA 209 206877_at−2.41 NM_002357 MAD 210 216565_x_at −2.35 AL121994 — 211 201363_s_at−2.38 AB020657 IVNS1ABP 212 205193_at −2.15 NM_012323 MAFF 213200648_s_at −2.2 NM_002065 GLUL 214 210513_s_at −2.46 AF091352 VEGF 215212481_s_at −2.11 AI214061 TPM4 216 211889_x_at −2.35 D12502 CEACAM1 217208991_at −2.26 AA634272 STAT3 218 212902_at −2.41 BE645231 SEC24A 219217414_x_at −2.44 V00489 — 220 212974_at −2.35 AI808958 KIAA0870 221202872_at −2.27 AW024925 ATP6V1C1 222 219540_at −2.28 AU150728 ZNF267223 217202_s_at −2.21 U08626 — 224 215838_at −1.86 AF212842 LIR9 225203085_s_at −2.16 BC000125 TGFB1 226 201329_s_at −2.42 NM_005239 ETS2227 217871_s_at −2.8 NM_002415 MIF 228 205323_s_at −2.19 NM_005955 MTF1229 205409_at −2.11 NM_005253 FOSL2 230 209795_at −2.89 L07555 CD69 231201846_s_at −2.17 NM_012234 RYBP 232 211250_s_at −2.71 AB000463 SH3BP2233 211368_s_at −2.53 U13700 CASP1 234 212226_s_at −2.83 AA628586 PPAP2B235 202022_at −2.39 NM_005165 ALDOC 236 200799_at −2.69 NM_005345 HSPA1A237 204490_s_at −2.27 M24915 CD44 238 212722_s_at −2.18 AK021780 PTDSR239 204435_at −2.24 NM_014778 NUPL1 240 212203_x_at −2.05 BF338947IFITM3 241 211506_s_at −1.99 AF043337 — 242 203927_at −2.91 NM_004556NFKBIE 243 207492_at −2.51 NM_025105 NGLY1 244 205633_s_at −2.52NM_000688 ALAS1 245 212252_at −2.07 AA181179 CAMKK2 246 215783_s_at−2.68 X14174 ALPL 247 208928_at −2.28 AF258341 POR 248 203175_at −2.27NM_001665 ARHG 249 217985_s_at −2.47 AA102574 BAZ1A 250 200664_s_at −2.6BG537255 DNAJB1 251 211429_s_at −2.39 AF119873 — 252 201411_s_at −2.36NM_017958 PLEKHB2 253 200075_s_at −2.44 BC006249 GUK1 254 201471_s_at−2.42 NM_003900 SQSTM1 255 208751_at −2.54 BC001165 NAPA 256 201315_x_at−1.82 NM_006435 IFITM2 257 220935_s_at −2.37 NM_018249 CDK5RAP2 258215719_x_at −1.64 X83493 TNFRSF6 259 206011_at −2.55 AI719655 CASP1 260213817_at −2.64 AL049435 — 261 202205_at −2.08 NM_003370 VASP 262211699_x_at −2.11 AF349571 HBA1 263 212761_at −2.11 AI949687 TCF7L2 264200998_s_at −2.33 AW029619 CKAP4 265 209116_x_at −2.3 M25079 HBB 266218881_s_at −2.1 NM_024530 FLJ23306 267 209354_at −2.13 BC002794TNFRSF14 268 205367_at −2.66 NM_020979 APS 269 208438_s_at −2.18NM_005248 FGR 270 209545_s_at −2.23 AF064824 RIPK2 271 208436_s_at −2.42NM_004030 IRF7 272 209761_s_at −2.26 AA969194 SP110 273 220330_s_at−2.46 NM_022136 SAMSN1 274 200632_s_at −2.22 NM_006096 NDRG1 275200822_x_at −2.67 NM_000365 TPI1 276 209835_x_at −2.22 BC004372 CD44 277204018_x_at −2.33 NM_000558 HBA1 278 38269_at −2.19 AL050147 PRKD2 279216252_x_at −1.55 Z70519 TNFRSF6 280 219434_at −1.85 NM_018643 TREM1 281218611_at −2.32 NM_016545 IER5 282 214211_at −2.41 AA083483 FTH1 283216316_x_at −2 X78713 — 284 218978_s_at −2.34 NM_018586 MSCP 285214084_x_at −2.41 AW072388 — 286 209446_s_at −2.6 BC001743 FLJ10803 287209939_x_at −2.08 AF005775 CFLAR 288 210789_x_at −2.28 L00692 CEACAM3289 204265_s_at −2.28 NM_022107 C6orf9 290 204166_at −2.28 NM_014963KIAA0963 291 219210_s_at −2.18 NM_016530 LOC51762 292 202545_at −2.19NM_006254 PRKCD 293 211696_x_at −1.98 AF349114 HBB 294 218088_s_at −2.66NM_022157 RRAGC 295 206911_at −2.07 NM_005082 ZNF147 296 204308_s_at−2.48 NM_014844 KIAA0329 297 200649_at −2.45 BC002356 NUCB1 298213011_s_at −2.39 BF116254 TPI1 299 202719_s_at −2.06 BC001451 TES 30041387_r_at −2.04 AB002344 KIAA0346 301 205026_at −1.85 NM_012448 STAT5B302 221484_at −1.97 BF691447 B4GALT5 303 205382_s_at −1.86 NM_001928 DF304 209046_s_at −2.22 AB030710 GABARAPL2 305 204924_at −2.13 NM_003264TLR2 306 207677_s_at −2.22 NM_013416 NCF4 307 203508_at −2.29 NM_001066TNFRSF1B 308 206245_s_at −2.38 NM_006469 IVNS1ABP 309 219947_at −2.15NM_016184 CLECSF6 310 209930_s_at −2.48 L13974 NFE2 311 219202_at −2.32NM_024599 FLJ22341 312 204907_s_at −2.17 AI829875 BCL3 313 208610_s_at−2.04 AI655799 SRRM2 314 211661_x_at −1.84 M80436 PTAFR 315 201762_s_at−2.06 NM_002818 PSME2 316 211302_s_at −1.85 L20966 PDE4B 317 212014_x_at−2.13 AI493245 CD44 318 219357_at −2.06 NM_014027 GTPBP1 319 203006_at−2.09 NM_005539 INPP5A 320 202833_s_at −2.14 NM_000295 SERPINA1 321203535_at −1.68 NM_002965 S100A9 322 202856_s_at −2.12 NM_004207 SLC16A3323 36564_at −1.92 W27419 FLJ90005 324 213988_s_at −2.09 BE971383 SAT325 216915_s_at −1.95 S69182 PTPN12 326 201192_s_at −1.96 NM_006224PITPN 327 209457_at −1.97 U16996 DUSP5 328 211806_s_at −2.27 D87291KCNJ15 329 204747_at −1.95 NM_001549 IFIT4 330 219209_at −2.13 NM_022168MDA5 331 215101_s_at −1.76 BG166705 CXCL5 332 201818_at −2.03 NM_024830FLJ12443 333 219082_at −2.71 NM_015944 CGI-14 334 208918_s_at −2.01AI334128 FLJ13052 335 200646_s_at −1.96 NM_006184 NUCB1 336 204157_s_at−1.95 NM_025164 KIAA0999 337 209906_at −2.02 U62027 C3AR1 338214511_x_at −1.79 L03419 FCGR1A 339 204489_s_at −2 NM_000610 CD44 340214486_x_at −2.14 AF041459 CFLAR 341 218660_at −1.96 NM_003494 DYSF 342209933_s_at −2.04 AF020314 CMRF-35H 343 203708_at −1.73 NM_002600 PDE4B344 209355_s_at −2.22 AB000889 PPAP2B 345 214121_x_at −1.95 AA086229ENIGMA 346 201942_s_at −2.25 D85390 CPD 347 207500_at −2.13 NM_004347CASP5 348 219593_at −2 NM_016582 SLC15A3 349 212171_x_at −2.02 H95344VEGF 350 211317_s_at −1.84 AF041461 CFLAR 351 208785_s_at −1.95 BE893893— 352 217078_s_at −2.02 AJ010102 CMRF-35H 353 208637_x_at −1.96 BC003576ACTN1 354 221524_s_at −1.78 AF272036 RRAGD 355 210564_x_at −1.85AF009619 CFLAR 356 209508_x_at −2.12 AF005774 CFLAR 357 203233_at −1.74NM_000418 IL4R 358 202861_at −1.92 NM_002616 PER1 359 205033_s_at −2.17NM_004084 DEFA1 360 206995_x_at −1.85 NM_003693 SCARF1 361 221985_at−1.89 AW006750 FLJ20059 362 202626_s_at −1.92 NM_002350 LYN 363220404_at −2.42 NM_014076 GPR97 364 216950_s_at −2.09 X14355 FCGR1A 365209117_at −1.93 U79458 WBP2 366 203591_s_at −1.66 NM_000760 CSF3R 367211275_s_at −1.56 AF087942 GYG 368 221827_at −2.17 BE788439 C20orf18 369213418_at −1.93 NM_002155 HSPA6 370 221764_at −1.87 AL574186 MGC16353371 214472_at −2.08 NM_003530 HIST1H3D 372 210629_x_at −2.05 AF000425LST1 373 206082_at −1.85 NM_006674 HCP5 374 209970_x_at −1.98 M87507CASP1 375 202093_s_at −1.89 NM_019088 PD2 376 208748_s_at −1.65 AA507012FLOT1 377 202509_s_at −1.85 AI862445 TNFAIP2 378 207674_at −1.52NM_002000 FCAR 379 217209_at −1.85 X16454 — 380 204581_at −2.48NM_001771 CD22 381 206278_at −1.67 D10202 PTAFR 382 205147_x_at −2.26NM_000631 NCF4 383 209193_at −1.86 M24779 PIM1 384 201783_s_at −1.86NM_021975 RELA 385 212496_s_at −1.98 BE256900 KIAA0876 386 209020_at−1.73 AF217514 C20orf111 387 212860_at −1.78 BG168720 ZDHHC18 38832069_at −1.66 AB014515 N4BP1 389 214792_x_at −2.18 AI955119 VAMP2 390200629_at −1.91 NM_004184 WARS 391 211012_s_at −1.93 BC000080 PML 392204053_x_at −2.04 U96180 PTEN 393 203925_at −2.04 NM_002061 GCLM 394215499_at −1.92 AA780381 MAP2K3 395 211862_x_at −1.98 AF015451 CFLAR 396214014_at −1.8 W81196 CDC42EP2 397 219774_at −1.67 NM_019044 FLJ10996398 200852_x_at −1.9 NM_005273 GNB2 399 202146_at −1.88 AA747426 IFRD1400 201844_s_at −1.79 W84482 RYBP 401 200744_s_at −1.91 AI741124 GNB1402 210190_at −1.61 AF071504 STX11 403 208052_x_at −1.87 NM_001815CEACAM3 404 203904_x_at −1.82 NM_002231 KAI1 405 31845_at −1.82 U32645ELF4 406 203194_s_at −1.94 AA527238 NUP98 407 212268_at −1.84 NM_030666SERPINB1 408 202842_s_at −1.98 AL080081 DNAJB9 409 206359_at −1.96BG035761 SOCS3 410 202934_at −1.83 AI761561 HK2 411 211307_s_at −1.56U43677 FCAR 412 204232_at −1.99 NM_004106 FCER1G 413 202769_at −1.89AW134535 CCNG2 414 210449_x_at −1.71 AF100544 MAPK14 415 200924_s_at−2.23 NM_002394 SLC3A2 416 220319_s_at −1.73 NM_013262 MIR 417 210701_at−1.99 D85939 CFDP1 418 204781_s_at −2.01 NM_000043 TNFRSF6 419208934_s_at −2.25 AF342815 LGALS8 420 210029_at −1.73 M34455 INDO 421215485_s_at −1.71 AA284705 ICAM1 422 221385_s_at −1.7 NM_005305 GPR42423 214681_at −2.08 AI830490 GK 424 201353_s_at −1.89 AI653126 BAZ2A 425213138_at −1.81 M62324 MRF-1 426 219911_s_at −1.84 NM_016354 SLC21A12427 212007_at −1.97 AI927512 UBXD2 428 201841_s_at −1.91 NM_001540 HSPB1429 210916_s_at −1.93 AF098641 CD44 430 205159_at −2.15 AV756141 CSF2RB431 205119_s_at −1.55 NM_002029 FPR1 432 203882_at −1.85 NM_006084ISGF3G 433 212041_at −1.97 AL566172 ATP6V0D1 434 203574_at −1.99NM_005384 NFIL3 435 205483_s_at −2.4 NM_005101 G1P2 436 204192_at −2.01NM_001774 CD37 437 219742_at −1.58 NM_030567 MGC10772 438 205312_at −2NM_003120 SPI1 439 211582_x_at −1.94 AF000424 LST1 440 200898_s_at −1.88AK002091 MGEA5 441 211366_x_at −2 U13698 CASP1 442 205965_at −1.78NM_006399 BATF 443 201625_s_at −1.92 BE300521 INSIG1 444 201303_at −1.63NM_014740 DDX48 445 200966_x_at −1.93 NM_000034 ALDOA 446 201400_at−1.88 NM_002795 PSMB3 447 206567_s_at −1.71 NM_016436 C20orf104 448211561_x_at −1.69 L35253 MAPK14 449 218855_at −2.25 NM_016372 TPRA40 450201531_at −2.09 NM_003407 ZFP36 451 202150_s_at −1.71 U64317 NEDD9 452211968_s_at −1.53 AI962933 HSPCA 453 221616_s_at −1.76 AF077053 TAF9L454 209383_at −1.73 BC003637 MARS 455 117_at −1.78 X51757cds HSPA6 456213445_at −1.73 D63484 KIAA0150 457 202671_s_at −2.33 NM_003681 PDXK 458209791_at −1.9 AL049569 PADI2 459 207667_s_at −1.8 NM_002756 MAP2K3 460208864_s_at −1.65 AF313911 TXN 461 211367_s_at −2 U13699 CASP1 462211160_x_at −1.88 M95178 ACTN1 463 217232_x_at −1.61 AF059180 — 464221978_at −1.73 BE138825 HLA-F 465 213593_s_at −2.03 AW978896 TRA2A 466213607_x_at −1.75 BE551347 — 467 207275_s_at −1.5 NM_001995 FACL2 468202708_s_at −2.01 NM_003528 HIST2H2BE 469 204095_s_at −1.62 AL521391 ELL470 202181_at −2.01 NM_014734 KIAA0247 471 202241_at −1.79 NM_025195C8FW 472 219257_s_at −2.19 NM_021972 SPHK1 473 218943_s_at −1.9NM_014314 RIG-I 474 214847_s_at −1.93 BG111168 C6orf9 475 208499_s_at−1.58 NM_006260 DNAJC3 476 201328_at −1.79 AL575509 ETS2 477 219952_s_at−1.77 NM_020533 MCOLN1 478 200730_s_at −1.65 BF576710 PTP4A1 479206491_s_at −1.73 NM_003827 NAPA 480 219403_s_at −1.88 NM_006665 HPSE481 218154_at −1.61 NM_024736 FLJ12150 482 215633_x_at −1.84 AV713720LST1 483 217492_s_at −1.87 AF023139 PTENP1 484 219259_at −1.76 NM_022367FLJ12287 485 209272_at −1.81 AF045451 NAB1 486 218404_at −2.05 NM_013322SNX10 487 220066_at −1.8 NM_022162 CARD15 488 218673_s_at −1.75NM_006395 GSA7 489 219066_at −1.7 NM_021823 MDS018 490 205160_s_at −1.85NM_001109 ADAM8 491 219359_at −1.9 NM_025092 FLJ22635 492 201132_at−1.77 NM_019597 HNRPH2 493 202855_s_at −1.86 AL513917 SLC16A3 494200766_at −1.92 NM_001909 CTSD 495 204970_s_at −1.59 NM_002359 MAFG 496212769_at −1.95 AI567426 TLE3 497 203922_s_at −1.93 AI308863 CYBB 498201670_s_at −1.81 M68956 MARCKS 499 221962_s_at −1.7 AI829920 UBE2H 500200704_at −1.56 AB034747 LITAF 501 209287_s_at −1.84 AF104857 CDC42EP3502 207113_s_at −1.66 NM_000594 TNF 503 210706_s_at −2 BC000213 RNF24504 221755_at −1.99 BG334196 DKFZp762C186 505 203692_s_at −1.68 AI640363E2F3 506 206472_s_at −1.92 NM_005078 TLE3 507 214574_x_at −1.73NM_007161 LST1 508 205844_at −2.09 NM_004666 VNN1 509 214687_x_at −1.91AK026577 ALDOA 510 203113_s_at −1.63 NM_001960 EEF1D 511 205349_at −1.78NM_002068 GNA15 512 214017_s_at −1.82 AA039439 DHX34 513 214911_s_at−1.71 S78771 BRD2 514 221617_at −1.85 AF077053 TAF9L 515 202910_s_at−1.81 NM_001784 CD97 516 202140_s_at −1.67 NM_003992 CLK3 517 203388_at−1.6 NM_004313 ARRB2 518 213112_s_at −1.82 N30649 SQSTM1 519 217966_s_at−1.78 NM_022083 C1orf24 520 204269_at −1.64 NM_006875 PIM2 521 206004_at−1.56 NM_003245 TGM3 522 219690_at −1.77 NM_024660 FLJ22573 523201943_s_at −1.89 NM_001304 CPD 524 216457_s_at −1.58 AK026080 SF3A1 525208485_x_at −1.87 NM_003879 CFLAR 526 201168_x_at −2 NM_004309 ARHGDIA527 35254_at −1.71 AB007447 FLN29 528 203394_s_at −1.55 BE973687 HES1529 210773_s_at −1.82 U81501 FPRL1 530 206576_s_at −1.72 NM_001712CEACAM1 531 212680_x_at −1.7 BE305165 PPP1R14B 532 200014_s_at −1.69NM_004500 HNRPC 533 218302_at −1.79 NM_018468 PEN2 534 214268_s_at −1.89AL042220 MTMR4 535 221571_at −1.76 AI721219 TRAF3 536 218136_s_at −1.81NM_018579 MSCP 537 209179_s_at −1.72 BC003164 LENG4 538 202446_s_at−1.69 AI825926 PLSCR1 539 200706_s_at −1.75 NM_004862 LITAF 540 91703_at−1.95 AA149545 DKFZp762C186 541 204493_at −1.61 NM_001196 BID 542203885_at −1.74 NM_014999 RAB21 543 211133_x_at −1.65 AF009643 LILRB3544 211014_s_at −1.77 AF230410 PML 545 222024_s_at −1.85 AK022014 AKAP13546 205945_at −1.83 NM_000565 IL6R 547 214181_x_at −1.77 AI735692 NCR3548 200905_x_at −1.59 NM_005516 HLA-E 549 211711_s_at −1.7 BC005821 PTEN550 210140_at −1.7 AF031824 CST7 551 200733_s_at −1.56 U48296 PTP4A1 552215975_x_at −1.74 X68285 GK 553 206571_s_at −1.83 NM_004834 MAP4K4 554209417_s_at −1.69 BC001356 IFI35 555 211135_x_at −1.72 AF009644 LILRB3556 204225_at −1.99 NM_006037 MGC16025 557 211013_x_at −1.61 AF230411PML 558 214508_x_at −1.57 U44836 CREM 559 221704_s_at −1.63 BC005882FLJ12750 560 203419_at −1.84 NM_014727 MLL4 561 211581_x_at −1.74AF000426 LST1 562 211816_x_at −1.51 D87858 FCAR 563 215645_at −1.54AF090883 FLJ11286 564 209967_s_at −1.71 D14826 CREM 565 200065_s_at−1.48 AF052179 ARF1 566 210225_x_at −1.69 AF009635 LILRB3 567205068_s_at −1.79 BE671084 GRAF 568 210772_at −1.76 M88107 FPRL1 569204769_s_at −1.63 M74447 TAP2 570 217868_s_at −1.67 NM_016025 DREV1 571217817_at −1.95 BE891920 ARPC4 572 211037_s_at −1.78 BC006309 LENG4 573215111_s_at −1.66 AK027071 TSC22 574 221882_s_at −1.69 AI636233 TMEM8575 40420_at −1.79 AB015718 STK10 576 203693_s_at −1.81 NM_001949 E2F3577 201926_s_at −1.33 BC001288 DAF 578 210610_at −1.59 M69176 CEACAM1579 218438_s_at −1.63 NM_025205 EG1 580 200971_s_at −1.61 NM_014445SERP1 581 205645_at −1.57 NM_004726 REPS2 582 204121_at −1.67 NM_006705GADD45G 583 206707_x_at −1.66 NM_015864 C6orf32 584 209919_x_at −1.79L20490 GGT1 585 207842_s_at −1.68 NM_007359 MLN51 586 215148_s_at −1.73AI141541 APBA3 587 217941_s_at −1.76 NM_018695 ERBB2IP 588 204858_s_at−1.63 NM_001953 ECGF1 589 211716_x_at −1.9 BC005851 ARHGDIA 590210784_x_at −1.56 AF009634 LILRB3 591 AFFX- −1.38 AFFX- — HUMISGF3A/HUMISGF3A/ M97935_5_at M97935_5 592 201642_at −1.72 NM_005534 IFNGR2 593209216_at −1.59 BC000464 JM5 594 201713_s_at −1.61 D42063 RANBP2 595211417_x_at −1.7 L20493 GGT1 596 203616_at −1.62 NM_002690 POLB 597202081_at −1.7 NM_004907 ETR101 598 201963_at −1.66 NM_021122 FACL2 599214618_at −1.62 AF015452 CFLAR 600 209695_at −1.58 BC003105 PTP4A3 601210233_at −1.62 AF167343 IL1RAP 602 202874_s_at −2.31 NM_001695 ATP6V1C1603 205627_at −1.56 NM_001785 CDA 604 214446_at −1.75 NM_012081 ELL2 60536994_at −1.76 M62762 ATP6V0C 606 207338_s_at −1.6 NM_003454 ZNF200 607203528_at −1.7 NM_006378 SEMA4D 608 220023_at −1.83 NM_018690 APOB48R609 205146_x_at −1.64 NM_004886 APBA3 610 218310_at −1.7 NM_014504RABGEF1 611 208012_x_at −1.83 NM_004509 SP110 612 203652_at −1.6NM_002419 MAP3K11 613 203749_s_at −1.59 AI806984 RARA 614 219862_s_at−1.86 NM_012336 NARF 615 214465_at −1.55 NM_000608 ORM1 616 209762_x_at−1.7 AF280094 SP110 617 202393_s_at −1.86 NM_005655 TIEG 618 211764_s_at−1.86 BC005980 UBE2D1 619 204794_at −1.74 NM_004418 DUSP2 620212359_s_at −1.58 W89120 KIAA0913 621 206513_at −1.67 NM_004833 AIM2 622221485_at −1.57 AL035683 B4GALT5 623 219806_s_at −1.65 NM_020179 FN5 624212457_at −1.57 AL161985 TFE3 625 211763_s_at −1.6 BC005979 UBE2B 626209882_at −1.69 AF084462 RIT1 627 202441_at −1.58 AL568449 KEO4 628201750_s_at −1.61 NM_001397 ECE1 629 218586_at −1.54 NM_018270 C20orf20630 209850_s_at −1.54 BC005406 CDC42EP2 631 201573_s_at −1.81 M75715ETF1 632 205425_at −1.88 NM_005338 HIP1 633 221653_x_at −1.57 BC004395APOL2 634 213603_s_at −1.63 BE138888 RAC2 635 203276_at −1.79 NM_005573LMNB1 636 205099_s_at −1.7 NM_001295 CCR1 637 202941_at −1.6 NM_021074NDUFV2 638 202082_s_at −1.59 AV748469 SEC14L1 639 203964_at −1.96NM_004688 NMI 640 202618_s_at −1.65 L37298 MECP2 641 201463_s_at −1.6NM_006755 TALDO1 642 208284_x_at −1.7 NM_013421 GGT1 643 219394_at −1.71NM_024419 PGS1 644 215603_x_at −1.47 AI344075 — 645 200656_s_at −1.74NM_000918 P4HB 646 212368_at −1.83 AA972711 ZNF292 647 202625_at −1.72AI356412 LYN 648 204243_at −1.79 NM_012421 RLF 649 205207_at −1.6NM_000600 IL6 650 209575_at −1.61 BC001903 IL10RB 651 217858_s_at −1.58NM_016607 ALEX3 652 214394_x_at −1.56 AI613383 EEF1D 653 213338_at −1.7BF062629 RIS1 654 217835_x_at −1.57 NM_018840 C20orf24 655 203140_at−1.75 NM_001706 BCL6 656 220470_at −1.48 NM_016526 BET1L 657 200954_at−1.67 NM_001694 ATP6V0C 658 207339_s_at −1.73 NM_002341 LTB 659222025_s_at −1.62 AI991887 OPLAH 660 206239_s_at −1.47 NM_003122 SPINK1661 209451_at −1.66 U59863 TANK 662 218624_s_at −1.58 NM_023939 MGC2752663 220104_at −1.56 NM_020119 ZAP 664 207072_at −1.41 NM_003853 IL18RAP665 211507_s_at −1.57 AF233437 MTMR3 666 208881_x_at −1.53 BC005247 IDI1667 221680_s_at −1.68 AF147782 ETV7 668 207075_at −1.46 NM_004895 CIAS1669 55705_at −1.69 W07773 MGC16353 670 215732_s_at −1.54 AK023924 DTX2671 214737_x_at −1.46 AV725195 HNRPC 672 206618_at −1.33 NM_003855IL18R1 673 218279_s_at −1.46 BC001629 — 674 205568_at −1.46 NM_020980AQP9 675 218255_s_at −1.58 NM_022452 FBS1 676 200881_s_at −1.71NM_001539 DNAJA1 677 207131_x_at −1.79 NM_013430 GGT1 678 214753_at−1.72 AW084068 CG005 679 212626_x_at −1.43 AA664258 HNRPC 680208967_s_at −1.52 U39945 AK2 681 209034_at −1.66 AF279899 PNRC1 682204958_at −1.52 NM_004073 CNK 683 218963_s_at −1.84 NM_015515 KRT23 684206503_x_at −1.55 NM_002675 PML 685 222175_s_at −1.55 AK000003 PCQAP 686200814_at −1.67 NM_006263 PSME1 687 202333_s_at −1.69 AA877765 UBE2B 688201921_at −1.64 NM_004125 GNG10 689 202083_s_at −1.53 AI017770 SEC14L1690 201693_s_at −1.75 AV733950 EGR1 691 202537_s_at −1.59 AF151842DKFZP564O123 692 215884_s_at −1.78 AK001029 — 693 209546_s_at −1.53AF323540 APOL1 694 202841_x_at −1.66 NM_007346 OGFR 695 209238_at −1.59BE966922 STX3A 696 214541_s_at −1.59 AF142418 QKI 697 203370_s_at −1.81NM_005451 ENIGMA 698 222150_s_at −1.53 AK026747 LOC54103 699 202382_s_at−1.68 NM_005471 GNPI 700 213142_x_at −1.39 AV700415 LOC54103 701213778_x_at −1.56 AI983201 FANCA 702 212689_s_at −1.61 AA524505 JMJD1703 201410_at −1.68 AI983043 PLEKHB2 704 210118_s_at −1.46 M15329 IL1A705 204601_at −1.3 NM_014664 N4BP1 706 220956_s_at −1.54 NM_017555 EGLN2707 218085_at −1.67 NM_015961 HSPC177 708 217591_at −1.58 BF725121 SKIL709 203109_at −1.59 NM_003969 UBE2M 710 210362_x_at −1.64 AF230409 PML711 202149_at −1.57 AL136139 NEDD9 712 203853_s_at −1.45 NM_012296 GAB2713 212443_at −1.68 AB011112 KIAA0540 714 202659_at −1.44 NM_002801PSMB10 715 207387_s_at −1.18 NM_000167 GK 716 204348_s_at −1.52NM_013410 AK3 717 207777_s_at −1.54 NM_007237 SP140 718 200904_at −1.46X56841 HLA-E 719 217986_s_at −1.52 NM_013448 BAZ1A 720 201940_at −1.6AA897514 CPD 721 202684_s_at −1.54 AB020966 RNMT 722 204929_s_at −1.66NM_006634 VAMP5 723 202859_x_at −1.4 NM_000584 IL8 724 203047_at −1.71NM_005990 STK10 725 210443_x_at −1.59 AF172452 OGFR 726 211799_x_at−1.52 U62824 HLA-C 727 207630_s_at −1.62 NM_001881 CREM 728 213596_at−1.63 AL050391 CASP4 729 201556_s_at −1.61 BC002737 VAMP2 730201296_s_at −1.72 NM_015626 WSB1 731 211865_s_at −1.52 AB013463 FZR1 732212561_at −1.57 AA349595 RAB6IP1 733 204099_at −1.56 NM_003078 SMARCD3734 203530_s_at −1.63 NM_004604 STX4A 735 217436_x_at −1.53 M80469 — 736205266_at −1.53 NM_002309 LIF 737 203028_s_at −1.69 NM_000101 CYBA 738206685_x_at −1.65 AA902767 BRD2 739 219183_s_at −1.7 NM_013385 PSCD4 740206247_at −1.61 NM_005931 MICB 741 217152_at −1.5 AK024136 NCOR1 742217962_at −1.57 NM_018648 NOLA3 743 214919_s_at −1.48 R39094 — 744200645_at −1.66 NM_007278 GABARAP 745 222326_at −1.24 AW973834 — 746207713_s_at −1.55 NM_006462 C20orf18 747 202688_at −1.78 NM_003810TNFSF10 748 201748_s_at −1.46 NM_002967 SAFB 749 219859_at −1.61NM_014358 CLECSF9 750 218130_at −1.57 NM_024510 MGC4368 751 200078_s_at−1.55 BC005876 ATP6V0B 752 200808_s_at −1.85 NM_003461 ZYX 753201170_s_at −1.74 NM_003670 BHLHB2 754 204158_s_at −1.4 NM_006019 TCIRG1755 202574_s_at −1.55 NM_001319 CSNK1G2 756 210754_s_at −1.65 M79321 LYN757 209270_at −1.4 L25541 LAMB3 758 204806_x_at −1.52 NM_018950 HLA-F759 204615_x_at −1.71 NM_004508 IDI1 760 209206_at −1.56 AV701283SEC22L1 761 203271_s_at −1.54 NM_005148 UNC119 762 209398_at −1.37BC002649 HIST1H1C 763 218319_at −1.39 NM_020651 PELI1 764 213191_at−1.42 AF070530 TRIF 765 211969_at −1.47 BG420237 HSPCA 766 205003_at−1.53 NM_014705 DOCK4 767 217422_s_at −1.65 X52785 CD22 768 217911_s_at−1.54 NM_004281 BAG3 769 209239_at −1.55 M55643 NFKB1 770 209912_s_at−1.56 AI373854 KIAA0415 771 204668_at −1.36 AL031670 RNF24 772208018_s_at −1.68 NM_002110 HCK 773 220467_at −1.47 NM_025032 FLJ21272774 220603_s_at −1.35 NM_018349 FLJ11175 775 210538_s_at −1.64 U37546BIRC3 776 202734_at −1.9 NM_004240 TRIP10 777 205585_at −1.6 NM_001987ETV6 778 201560_at −1.42 NM_013943 CLIC4 779 205715_at −1.4 NM_004334BST1 780 221905_at −1.56 BF516433 CYLD 781 202255_s_at −1.49 NM_015556KIAA0440 782 201941_at −1.69 BE349147 CPD 783 214965_at −1.52 AF070574MGC26885 784 206758_at −1.68 NM_019886 CHST7 785 204507_s_at −1.61NM_000945 PPP3R1 786 214083_at −1.48 AW772123 PPP2R5C 787 212540_at−1.56 BG476661 CDC34 788 219763_at −1.62 NM_024820 KIAA1608 789221223_x_at −1.48 NM_013324 CISH 790 211605_s_at −1.65 U41742 RARA 791202018_s_at −1.51 NM_002343 LTF 792 206697_s_at −1.43 NM_005143 HP 793220941_s_at −1.38 NM_017447 C21orf91 794 212900_at −1.59 AJ131244 SEC24A795 209099_x_at −1.55 U73936 JAG1 796 214693_x_at −1.35 BE732345 NOTCH2797 204804_at −1.56 NM_003141 SSA1 798 202928_s_at −1.56 NM_024165 PHF1799 201186_at −1.57 NM_002337 LRPAP1 800 210873_x_at −1.65 U03891APOBEC3A 801 209192_x_at −1.49 BC000166 HTATIP 802 212242_at −1.64AL565074 TUBA1 803 202129_s_at −1.7 AW006290 RIOK3 804 210951_x_at −1.55AF125393 RAB27A 805 204118_at −1.63 NM_001778 CD48 806 36829_at −1.39AF022991 PER1 807 202391_at −1.47 NM_006317 BASP1 808 203490_at −1.5NM_001421 ELF4 809 200055_at −1.58 NM_006284 TAF10 810 210240_s_at −1.45U20498 CDKN2D 811 219492_at −1.67 NM_012110 CHIC2 812 211672_s_at −1.56AF019888 ARPC4 813 204293_at −1.56 NM_000199 SGSH 814 207992_s_at −1.7NM_000480 AMPD3 815 207419_s_at −1.6 NM_002872 RAC2 816 211702_s_at−1.51 AF350251 USP32 817 209477_at −1.52 BC000738 EMD 818 214590_s_at−1.41 AL545760 UBE2D1 819 218728_s_at −1.39 NM_014184 HSPC163 820202687_s_at −1.34 U57059 TNFSF10 821 212277_at −1.69 AB014547 MTMR4 822219625_s_at −1.65 NM_005713 COL4A3BP 823 221724_s_at −1.54 AF200738CLECSF6 824 212004_at −1.53 AL050028 DKFZp566C0424 825 212947_at −1.65AL031685 SLC9A8 826 212286_at −1.66 AW572909 KIAA0874 827 AFFX- −1.49AFFX- — HUMISGF3A/ HUMISGF3A/ M97935_MB_at M97935_MB 828 214866_at −1.48X74039 PLAUR

TABLE II Genes whose Expression is Downregulated in Septic as comparedto Aseptic Inflammation Affymetrix ® Fold Gene Rank U133A name Change“Source” symbol 1 202345_s_at 18.83 NM_001444 FABP5 2 201847_at 7.21NM_000235 LIPA 3 206488_s_at 10.7 NM_000072 CD36 4 201005_at 7.21NM_001769 CD9 5 201141_at 6.93 NM_002510 GPNMB 6 211734_s_at 4.17BC005912 FCER1A 7 211719_x_at 6.45 BC005858 FN1 8 216442_x_at 6.45AK026737 FN1 9 212192_at 4.85 AI718937 LOC115207 10 204787_at 5.16NM_007268 Z39IG 11 218559_s_at 3.83 NM_005461 MAFB 12 201212_at 5.7D55696 LGMN 13 209555_s_at 4.02 M98399 CD36 14 212464_s_at 5.3 X02761FN1 15 213872_at 6.62 BE465032 C6orf62 16 219607_s_at 6.17 NM_024021MS4A4A 17 210495_x_at 5.4 AF130095 FN1 18 208146_s_at 4.07 NM_031311CPVL 19 201279_s_at 2.97 BC003064 DAB2 20 211571_s_at 4.15 D32039 CSPG221 204112_s_at 4.1 NM_006895 HNMT 22 201278_at 3.64 N21202 — 23202437_s_at 3.45 NM_000104 CYP1B1 24 205695_at 4.13 NM_006843 SDS 25210757_x_at 2.46 AF188298 DAB2 26 203645_s_at 3.39 NM_004244 CD163 27212671_s_at 4.04 BG397856 HLA-DQA1 28 215049_x_at 3.36 Z22969 CD163 29201280_s_at 3.83 NM_001343 DAB2 30 201012_at 3.01 NM_000700 ANXA1 31202388_at 2.81 NM_002923 RGS2 32 215646_s_at 3.25 R94644 CSPG2 33213566_at 3.79 NM_005615 RNASE6 34 214770_at 4.69 AI299239 MSR1 35201273_s_at 3.14 NM_003133 SRP9 36 200762_at 3.59 NM_001386 DPYSL2 37209728_at 2.89 BC005312 HLA-DRB3 38 209377_s_at 3.56 AF274949 HMGN3 39200937_s_at 3.35 NM_000969 RPL5 40 214085_x_at 3.24 AI912583 HRB2 41201302_at 3.72 NM_001153 ANXA4 42 213619_at 2.63 AV753392 HNRPH1 43201947_s_at 3.55 NM_006431 CCT2 44 201324_at 3.65 NM_001423 EMP1 45211991_s_at 4.2 M27487 HLA-DPA1 46 201938_at 3.09 NM_004642 CDK2AP1 47209875_s_at 2.52 M83248 SPP1 48 218723_s_at 3.76 NM_014059 RGC32 49201034_at 3.06 BE545756 ADD3 50 202436_s_at 2.22 AU144855 CYP1B1 51219666_at 3.07 NM_022349 MS4A6A 52 213699_s_at 3.06 AA854017 — 53218150_at 2.64 NM_012097 ARL5 54 201339_s_at 4.05 NM_002979 SCP2 55203139_at 2.44 NM_004938 DAPK1 56 201360_at 3.08 NM_000099 CST3 57201413_at 2.34 NM_000414 HSD17B4 58 202591_s_at 3.62 NM_003143 SSBP1 59216834_at 2.92 S59049 RGS1 60 210338_s_at 3.38 AB034951 HSPA8 61221019_s_at 2.07 NM_030781 COLEC12 62 207761_s_at 2.97 NM_014033DKFZP586A0522 63 201068_s_at 3.31 NM_002803 PSMC2 64 202207_at 4.05BG435404 ARL7 65 211675_s_at 2.98 AF054589 HIC 66 211733_x_at 3.08BC005911 SCP2 67 211784_s_at 2.88 BC006181 SFRS1 68 202113_s_at 3.38AF043453 SNX2 69 204438_at 3.29 NM_002438 MRC1 70 201137_s_at 3.02NM_002121 HLA-DPB1 71 201301_s_at 3.36 BC000182 ANXA4 72 202546_at 2.83NM_003761 VAMP8 73 208638_at 3.67 BE910010 ATP6V1C2 74 208923_at 3.04BC005097 CYFIP1 75 200034_s_at 3.03 NM_000970 RPL6 76 202741_at 2.46AA130247 PRKACB 77 200703_at 3.23 NM_003746 DNCL1 78 218109_s_at 3.32NM_022736 FLJ14153 79 217478_s_at 2.84 X76775 HLA-DMA 80 213564_x_at3.01 BE042354 LDHB 81 221773_at 2.61 AW575374 ELK3 82 212586_at 2.93AA195244 CAST 83 200016_x_at 2.75 NM_002136 HNRPA1 84 211990_at 2.89M27487 HLA-DPA1 85 200657_at 2.98 NM_001152 SLC25A5 86 200036_s_at 3.04NM_007104 RPL10A 87 208627_s_at 3 BE966374 NSEP1 88 200893_at 2.7NM_004593 SFRS10 89 214560_at 2.07 NM_002030 FPRL2 90 213080_x_at 3.01BF214492 RPL5 91 200978_at 3.05 NM_005917 MDH1 92 200082_s_at 2.7AI805587 RPS7 93 205292_s_at 3.05 NM_002137 HNRPA2B1 94 202087_s_at 2.97NM_001912 CTSL 95 200926_at 2.64 NM_001025 RPS23 96 211972_x_at 3.11AI953822 RPLP0 97 201193_at 2.72 NM_005896 IDH1 98 200074_s_at 2.88U16738 RPL14 99 208981_at 2.33 AA702701 PECAM1 100 201944_at 2.23NM_000521 HEXB 101 204006_s_at 1.48 NM_000570 FCGR3A 102 221476_s_at 3.1AF279903 RPL15 103 215691_x_at 2.63 AV702994 — 104 209200_at 2.64AL536517 MEF2C 105 203741_s_at 2.53 NM_001114 ADCY7 106 201754_at 2.98NM_004374 COX6C 107 212907_at 2.79 AI972416 SLC30A1 108 208983_s_at 2.2M37780 PECAM1 109 217802_s_at 2.76 NM_022731 NUCKS 110 202605_at 2.9NM_000181 GUSB 111 211720_x_at 3.1 BC005863 RPLP0 112 203305_at 2.58NM_000129 F13A1 113 200063_s_at 2.64 BC002398 NPM1 114 201154_x_at 2.85NM_000968 RPL4 115 213738_s_at 2.91 AI587323 ATP5A1 116 202377_at 2.03AW026535 LEPR 117 211986_at 2.9 BG287862 MGC5395 118 207507_s_at 3.04NM_001689 ATP5G3 119 204057_at 1.81 AI073984 ICSBP1 120 201033_x_at 2.93NM_001002 RPLP0 121 201293_x_at 2.87 NM_021130 PPIA 122 207508_at 3.14NM_001689 ATP5G3 123 200705_s_at 2.97 NM_001959 EEF1B2 124 208818_s_at3.34 BC000419 COMT 125 217724_at 2.64 AF131807 PAI-RBP1 126 200681_at2.52 NM_006708 GLO1 127 200750_s_at 2.62 AF054183 RAN 128 201952_at 2.51AA156721 ALCAM 129 203104_at 2.85 NM_005211 CSF1R 130 219563_at 1.86NM_024633 C14orf139 131 213737_x_at 2.11 AI620911 — 132 208856_x_at 2.84BC003655 RPLP0 133 207168_s_at 2.17 NM_004893 H2AFY 134 213377_x_at 2.85AI799007 RPS12 135 221841_s_at 2.41 BF514079 KLF4 136 201406_at 2.55NM_021029 RPL36A 137 215091_s_at 2.7 BE542815 GTF3A 138 200033_at 2.23NM_004396 DDX5 139 213941_x_at 2.63 AI970731 RPS7 140 201506_at 2.69NM_000358 TGFBI 141 212426_s_at 2.65 BF033313 YWHAQ 142 202075_s_at 2.12NM_006227 PLTP 143 200736_s_at 2.3 NM_000581 GPX1 144 214141_x_at 2.84BF033354 SFRS7 145 213048_s_at 2.45 W26593 SET 146 211755_s_at 2.66BC005960 ATP5F1 147 204620_s_at 2.07 NM_004385 CSPG2 148 221210_s_at1.98 NM_030769 NPL 149 209191_at 2.99 BC002654 TUBB-5 150 206682_at 2.12NM_006344 CLECSF13 151 218203_at 2.7 NM_013338 ALG5 152 221802_s_at 2.06AU157109 KIAA1598 153 213356_x_at 2.61 AL568186 HNRPA1 154 201427_s_at2.22 NM_005410 SEPP1 155 221731_x_at 2.16 BF218922 CSPG2 156 213416_at2.01 BG532690 ITGA4 157 213979_s_at 2.33 BF984434 CTBP1 158 201586_s_at2.81 NM_005066 SFPQ 159 201398_s_at 2.64 BC000687 TRAM1 160 211710_x_at2.82 BC005817 RPL4 161 212296_at 2.82 NM_005805 PSMD14 162 214182_at2.12 AA243143 ARF6 163 201785_at 2.14 NM_002933 RNASE1 164 201049_s_at2.81 NM_022551 RPS18 165 212010_s_at 2.27 AK025647 H41 166 214167_s_at2.88 AA555113 — 167 208683_at 2.38 M23254 CAPN2 168 211072_x_at 2.66BC006481 K-ALPHA-1 169 209696_at 2.5 D26054 FBP1 170 219725_at 1.84NM_018965 TREM2 171 204222_s_at 2.41 NM_006851 GLIPR1 172 213655_at 2.08AA502643 YWHAE 173 210139_s_at 2.44 L03203 PMP22 174 201084_s_at 2.6NM_014739 BTF 175 209480_at 2.19 M16276 HLA-DQB1 176 200768_s_at 2.56BC001686 MAT2A 177 220547_s_at 2.68 NM_019054 MGC5560 178 217869_at 2.69NM_016142 HSD17B12 179 219279_at 2.06 NM_017718 DOCK10 180 213537_at2.32 AI128225 HLA-DPA1 181 200023_s_at 2.93 NM_003754 EIF3S5 182200015_s_at 2.15 NM_004404 NEDD5 183 203316_s_at 2.54 NM_003094 SNRPE184 200004_at 2.2 NM_001418 EIF4G2 185 215193_x_at 2.74 AJ297586HLA-DRB3 186 205898_at 2.5 U20350 CX3CR1 187 203799_at 2.6 NM_014880DCL-1 188 208697_s_at 2.39 BC000734 EIF3S6 189 211945_s_at 2.45 BG500301ITGB1 190 219358_s_at 2.59 NM_018404 CENTA2 191 210982_s_at 2.41 M60333HLA-DRA 192 200693_at 2.54 NM_006826 YWHAQ 193 218005_at 2.51 AA744771ZNF22 194 213801_x_at 2.54 AW304232 LAMR1 195 206028_s_at 2.24 NM_006343MERTK 196 217773_s_at 2.44 NM_002489 NDUFA4 197 208654_s_at 2.03BF669455 CD164 198 201909_at 2.24 NM_001008 RPS4Y 199 205055_at 1.89NM_002208 ITGAE 200 208073_x_at 2.61 NM_003316 TTC3 201 200981_x_at 1.97NM_016592 GNAS 202 218007_s_at 2.54 NM_015920 RPS27L 203 221452_s_at2.99 NM_030969 TMEM14B 204 202544_at 2.45 NM_004124 GMFB 205 219452_at1.9 NM_022355 DPEP2 206 211985_s_at 1.73 AI653730 CALM1 207 219032_x_at2.5 NM_014322 OPN3 208 211378_x_at 2.64 BC001224 PPIA 209 213047_x_at2.41 AI278616 SET 210 200081_s_at 2.45 BE741754 RPS6 211 201368_at 1.89U07802 — 212 210027_s_at 2.34 M80261 APEX1 213 211058_x_at 2.59 BC006379K-ALPHA-1 214 201338_x_at 2.57 NM_002097 GTF3A 215 201090_x_at 2.59NM_006082 K-ALPHA-1 216 201310_s_at 2.09 NM_004772 C5orf13 217210891_s_at 1.94 AF035737 GTF2I 218 208787_at 2.67 BC003375 MRPL3 219208669_s_at 2.75 AF109873 CRI1 220 218589_at 2.6 NM_005767 P2RY5 221212638_s_at 2.17 BF131791 WWP1 222 205882_x_at 2.34 AI818488 ADD3 223212188_at 2.44 AA551075 LOC115207 224 34210_at 2.38 N90866 CDW52 225200838_at 2.17 NM_001908 CTSB 226 201590_x_at 2.63 NM_004039 ANXA2 227201092_at 2.23 NM_002893 RBBP7 228 201153_s_at 2.27 NM_021038 MBNL1 229213646_x_at 2.44 BE300252 K-ALPHA-1 230 200605_s_at 2.35 NM_002734PRKAR1A 231 213503_x_at 2.53 BE908217 ANXA2 232 211765_x_at 2.73BC005982 PPIA 233 208628_s_at 2.72 BC002411 NSEP1 234 204959_at 2.65NM_002432 MNDA 235 201665_x_at 2.55 NM_001021 RPS17 236 201795_at 1.9NM_002296 LBR 237 212537_x_at 2.14 BE733979 RPL17 238 200723_s_at 2.45NM_005898 M11S1 239 210427_x_at 2.55 BC001388 ANXA2 240 209684_at 2.23AL136924 RIN2 241 219519_s_at 2.44 NM_023068 SN 242 203060_s_at 2.43AF074331 PAPSS2 243 200818_at 2.56 NM_001697 ATP5O 244 210895_s_at 2.29L25259 CD86 245 217757_at 2.08 NM_000014 A2M 246 212639_x_at 2.48AL581768 K-ALPHA-1 247 209031_at 1.51 AL519710 IGSF4 248 201268_at 2.34NM_002512 NME2 249 201300_s_at 2.2 NM_000311 PRNP 250 218200_s_at 2.54NM_004546 NDUFB2 251 200839_s_at 2.03 NM_001908 CTSB 252 200038_s_at2.22 NM_000985 RPL17 253 202838_at 2.51 NM_000147 FUCA1 254 201064_s_at2.63 NM_003819 PABPC4 255 220945_x_at 1.8 NM_018050 FLJ10298 256214548_x_at 1.62 AF064092 GNAS 257 211487_x_at 2.62 BC004886 — 258211858_x_at 1.88 AF088184 GNAS 259 219777_at 2.26 NM_024711 hIAN2 260213831_at 1.75 X00452 HLA-DQA1 261 200002_at 2.23 NM_007209 RPL35 262203381_s_at 1.53 N33009 APOE 263 200089_s_at 2.62 AI953886 RPL4 264212270_x_at 2.05 BG168283 RPL17 265 208870_x_at 2.28 BC000931 ATP5C1 266209199_s_at 2.21 N22468 MEF2C 267 209312_x_at 2.36 U65585 HLA-DRB3 268212298_at 1.52 BE620457 NRP1 269 215096_s_at 2.64 AU145746 ESD 270202232_s_at 2.42 NM_006360 GA17 271 200662_s_at 2.13 NM_014765 TOMM20-PENDING 272 208998_at 2.37 U94592 UCP2 273 208858_s_at 2.19 BC004998MBC2 274 201973_s_at 1.77 AL550875 — 275 214042_s_at 2.55 AW071997 — 27644790_s_at 2.08 AI129310 C13orf18 277 201753_s_at 2.14 NM_019903 ADD3278 200873_s_at 2.03 NM_006585 CCT8 279 200010_at 2.51 NM_000975 RPL11280 206991_s_at 2.29 NM_000579 CCR5 281 208687_x_at 2.64 AF352832 HSPA8282 202602_s_at 2.1 NM_014500 HTATSF1 283 212205_at 1.75 AA534860 H2AV284 211939_x_at 2.25 X74070 BTF3 285 212159_x_at 2.35 AI125280 AP2A2 286221775_x_at 2.51 BG152979 — 287 203663_s_at 2.29 NM_004255 COX5A 288211978_x_at 2.44 AI708767 PPIA 289 201197_at 2.35 NM_001634 AMD1 290213366_x_at 2.44 AV711183 ATP5C1 291 212661_x_at 2.49 BE731738 — 292200780_x_at 1.81 NM_000516 GNAS 293 206768_x_at 2.45 D17652 RPL22 294200088_x_at 2.45 AK026491 — 295 201185_at 2.38 NM_002775 PRSS11 296218467_at 2.25 NM_020232 HCCA3 297 211285_s_at 2.58 U84404 UBE3A 298200595_s_at 2.08 NM_003750 EIF3S10 299 203932_at 2.41 NM_002118 HLA-DMB300 212273_x_at 1.84 AI591100 GNAS 301 201200_at 1.95 NM_003851 CREG 302221691_x_at 2.35 AB042278 NPM1 303 201515_s_at 1.99 NM_004622 TSN 304212956_at 2.16 AI348094 KIAA0882 305 205711_x_at 2.47 NM_005174 ATP5C1306 218224_at 1.68 NM_006029 PNMA1 307 212248_at 1.95 AI886796 LYRIC 308208894_at 2.29 M60334 HLA-DRA 309 209067_s_at 2.33 D89092 HNRPDL 310221891_x_at 2.79 AA704004 HSPA8 311 208833_s_at 2.07 AF119662 E46L 312218139_s_at 2.25 NM_018229 C14orf108 313 201030_x_at 2.24 NM_002300 LDHB314 211747_s_at 2.19 BC005938 LSM5 315 208944_at 2.61 D50683 TGFBR2 316220890_s_at 1.83 NM_016355 DDX47 317 215450_at 2.26 W87901 SNRPE 318201520_s_at 2.6 BF034561 GRSF1 319 202206_at 2.76 AW450363 ARL7 320203382_s_at 1.62 NM_000041 APOE 321 215236_s_at 2.04 AV721177 PICALM 322214323_s_at 1.8 N36842 UPF3A 323 200651_at 2.37 NM_006098 GNB2L1 324217870_s_at 2.18 NM_016308 UMP-CMPK 325 200909_s_at 2.17 NM_001004 RPLP2326 200028_s_at 2.38 NM_020151 STARD7 327 212658_at 2.17 N66633 LHFPL2328 213347_x_at 2.3 AW132023 RPS4X 329 200061_s_at 2.48 BC000523 RPS24330 212131_at 2.02 BG054966 DKFZP434D1335 331 213588_x_at 2.44 AA838274RPL14 332 217740_x_at 2.3 NM_000972 RPL7A 333 218577_at 2.22 NM_017768FLJ20331 334 213044_at 2.28 N22548 ROCK1 335 201078_at 1.84 NM_004800TM9SF2 336 217873_at 2.14 NM_016289 MO25 337 208319_s_at 2.06 NM_006743RBM3 338 212386_at 1.99 BF592782 TCF4 339 204319_s_at 1.92 NM_002925RGS10 340 208674_x_at 2.51 BC002594 DDOST 341 214500_at 2.06 AF044286H2AFY 342 202266_s_at 1.15 J04152 TACSTD2 343 200099_s_at 2.47 AL356115— 344 200872_at 2.31 NM_002966 S100A10 345 221253_s_at 2.25 NM_030810TXNDC5 346 211656_x_at 2.16 M32577 HLA-DQB1 347 221760_at 2.2 BG287153MAN1A1 348 200910_at 2.36 NM_005998 CCT3 349 201330_at 2.17 NM_002887RARS 350 216274_s_at 2.07 N99438 SPC18 351 203153_at 2.12 NM_001548IFIT1 352 200858_s_at 2.4 NM_001012 RPS8 353 202679_at 2.24 NM_000271NPC1 354 209389_x_at 2.3 M15887 DBI 355 212406_s_at 2.06 AB028973 MYT1356 202283_at 1.68 NM_002615 SERPINF1 357 209251_x_at 2.22 BC004949TUBA6 358 201697_s_at 1.97 NM_001379 DNMT1 359 212830_at 1.87 W68084EGFL5 360 212578_x_at 2.31 BF026595 — 361 212856_at 2.16 AB018310KIAA0767 362 218090_s_at 2.21 NM_018117 WDR11 363 200911_s_at 1.73NM_006283 TACC1 364 211684_s_at 2.46 AF250307 DNCI2 365 200030_s_at 2.34NM_002635 SLC25A3 366 212250_at 2.06 AV700332 LYRIC 367 221844_x_at 2.23AV756161 — 368 200949_x_at 2.48 NM_001023 RPS20 369 200754_x_at 2.1NM_003016 SFRS2 370 213084_x_at 2.38 BF125158 RPL23A 371 211984_at 1.88AI653730 CALM1 372 200915_x_at 1.95 NM_004986 KTN1 373 202231_at 2.17NM_006360 GA17 374 212918_at 2.07 AI962943 FLJ22028 375 201129_at 2.42NM_006276 SFRS7 376 208758_at 2.07 D89976 ATIC 377 221434_s_at 2.37NM_031210 DC50 378 210024_s_at 2.41 AB017644 UBE2E3 379 208834_x_at 2.38BC001865 RPL23A 380 217900_at 2.18 NM_018060 FLJ10326 381 220495_s_at2.05 NM_024715 FLJ22625 382 211971_s_at 1.89 AI653608 LRPPRC 383220960_x_at 2.36 NM_000983 RPL22 384 219506_at 1.76 NM_024579 FLJ23221385 201112_s_at 2.15 NM_001316 CSE1L 386 212191_x_at 2.04 AW574664 RPL13387 209619_at 2.18 K01144 CD74 388 200728_at 1.89 BE566290 ACTR2 38935974_at 2.42 U10485 LRMP 390 214003_x_at 2.22 BF184532 RPS20 391200631_s_at 1.9 NM_003011 SET 392 209397_at 1.96 BC000147 ME2 393201028_s_at 2.2 U82164 CD99 394 204661_at 2.23 NM_001803 CDW52 395205987_at 1.35 NM_001765 CD1C 396 200809_x_at 2.32 NM_000976 RPL12 397217832_at 2.15 BE672181 NSAP1 398 209861_s_at 2.32 U13261 METAP2 399201923_at 2.17 NM_006406 PRDX4 400 212952_at 2.01 AA910371 CALR 401209823_x_at 2.28 M17955 HLA-DQB1 402 208825_x_at 2.37 U43701 RPL23A 403211750_x_at 2.09 BC005946 TUBA6 404 201619_at 1.74 NM_006793 PRDX3 405211666_x_at 2.38 L22453 RPL3 406 213135_at 1.92 U90902 TIAM1 407214709_s_at 1.92 Z22551 KTN1 408 204150_at 2.02 NM_015136 STAB1 409208517_x_at 1.94 NM_001207 BTF3 410 215182_x_at 1.39 AL050122 — 411203804_s_at 1.79 NM_006107 LUC7A 412 213274_s_at 1.69 AA020826 CTSB 413201403_s_at 2.25 NM_004528 MGST3 414 212482_at 1.85 BF671894 FLJ13910415 209974_s_at 1.91 AF047473 BUB3 416 208306_x_at 2.12 NM_021983HLA-DRB3 417 212371_at 1.9 AL049397 PNAS-4 418 212998_x_at 1.9 AI583173HLA-DQB2 419 200025_s_at 2.26 NM_000988 RPL27 420 203156_at 1.64NM_016248 AKAP11 421 208703_s_at 1.6 BG427393 APLP2 422 220526_s_at 2.38NM_017971 MRPL20 423 211779_x_at 2.11 BC006155 AP2A2 424 221748_s_at2.02 AL046979 TNS 425 221475_s_at 2.18 NM_002948 RPL15 426 212166_at2.14 AL514547 CPNE1 427 217906_at 2.02 NM_014315 KLHDC2 428 212036_s_at2.28 AW152664 PNN 429 204141_at 2.2 NM_001069 TUBB 430 203403_s_at 2.25NM_005977 RNF6 431 202396_at 1.62 NM_006706 TCERG1 432 200740_s_at 2.37NM_006936 SMT3H1 433 201784_s_at 1.97 NM_014267 SMAP 434 204616_at 2.35NM_006002 UCHL3 435 205466_s_at 2.02 NM_005114 HS3ST1 436 209154_at 1.85AF234997 TIP-1 437 202502_at 1.75 NM_000016 ACADM 438 216241_s_at 2.07X57198 TCEA1 439 211070_x_at 2.39 BC006466 DBI 440 201264_x_at 2.37NM_001010 RPS6 441 210889_s_at 1.89 M31933 FCGR2B 442 212904_at 2.12AB033011 KIAA1185 443 210645_s_at 2.1 D83077 TTC3 444 200963_x_at 1.97NM_000993 RPL31 445 213687_s_at 2.22 BE968801 RPL35A 446 216640_s_at2.05 AK026926 — 447 221565_s_at 1.83 BC000039 LOC51063 448 38487_at 1.79D87433 STAB1 449 204342_at 2.24 NM_013386 DKFZp586G0123 450 219471_at2.09 NM_025113 C13orf18 451 217719_at 2.22 NM_016091 EIF3S6IP 452209240_at 1.69 AF070560 OGT 453 210149_s_at 2.14 AF061735 ATP5H 454200008_s_at 1.93 D13988 GDI2 455 217491_x_at 2.07 AF042165 COX7C 456200834_s_at 1.97 NM_001024 RPS21 457 200933_x_at 2.37 NM_001007 RPS4X458 213642_at 1.75 BE312027 RPL27 459 208804_s_at 1.97 AL031681 SFRS6460 218263_s_at 1.83 NM_021211 LOC58486 461 208997_s_at 2.09 U82819 UCP2462 221726_at 1.9 BE250348 RPL22 463 211073_x_at 2.24 BC006483 RPL3 464206710_s_at 1.77 NM_012307 EPB41L3 465 200943_at 2.32 NM_004965 HMGN1466 201687_s_at 1.94 NM_006595 API5 467 201217_x_at 2.35 NM_000967 RPL3468 202088_at 1.97 AI635449 SLC39A6 469 201327_s_at 1.91 NM_001762 CCT6A470 207040_s_at 2.01 NM_003932 ST13 471 214830_at 1.99 AI537540 SLC38A6472 201312_s_at 2.16 NM_003022 SH3BGRL 473 202428_x_at 2.29 NM_020548DBI 474 218802_at 2.21 NM_017918 FLJ20647 475 203012_x_at 2.16 NM_000984RPL23A 476 216342_x_at 2.26 AL121916 — 477 203534_at 1.83 NM_014462 LSM1478 212585_at 2.75 BF970829 OSBPL8 479 209118_s_at 2.11 AF141347 TUBA3480 209092_s_at 2.25 AF061730 CGI-150 481 212888_at 1.7 BG109746 DICER1482 204670_x_at 2.12 NM_002125 HLA-DRB3 483 202077_at 2.13 NM_005003NDUFAB1 484 213864_s_at 1.69 AI985751 NAP1L1 485 201444_s_at 1.87NM_005765 ATP6AP2 486 208929_x_at 1.96 BC004954 RPL13 487 208775_at 1.51D89729 XPO1 488 200763_s_at 2.05 NM_001003 RPLP1 489 201946_s_at 2.21AL545982 CCT2 490 218041_x_at 1.97 NM_018573 SLC38A2 491 201325_s_at1.69 NM_001423 EMP1 492 217945_at 1.94 NM_025238 BTBD1 493 204619_s_at1.75 BF590263 CSPG2 494 217362_x_at 1.99 AF005487 HLA-DRB3 495 202351_at2.17 AI093579 ITGAV 496 202381_at 2.02 NM_003816 ADAM9 497 220044_x_at1.81 NM_016424 LUC7A 498 203547_at 2.24 U47924 CD4 499 201071_x_at 2.44NM_012433 SF3B1 500 206978_at 1.78 NM_000647 CCR2 501 211185_s_at 2.14AF130099 FLJ14753 502 208617_s_at 1.82 AF208850 PTP4A2 503 200726_at2.04 NM_002710 PPP1CC 504 208982_at 1.66 AW574504 PECAM1 505 212039_x_at2.37 BG339228 RPL3 506 200091_s_at 2.04 AA888388 RPS25 507 217846_at2.24 NM_005051 QARS 508 212560_at 2.16 AV728268 SORL1 509 203473_at 1.67NM_007256 SLC21A9 510 200955_at 2.14 NM_006839 IMMT 511 205988_at 1.68NM_003874 CD84 512 212266_s_at 1.81 AW084582 SFRS5 513 212515_s_at 2.24BG492602 DDX3X 514 219065_s_at 1.78 NM_015955 CGI-27 515 202469_s_at1.95 AU149367 CPSF6 516 221751_at 1.74 AL565516 PANK3 517 217933_s_at1.92 NM_015907 LAP3 518 203485_at 1.93 NM_021136 RTN1 519 218191_s_at2.23 NM_018368 FLJ11240 520 208635_x_at 1.88 BF976260 NACA 521204892_x_at 1.93 NM_001402 EEF1A1 522 203721_s_at 2.07 NM_016001 CGI-48523 212640_at 1.8 AV712602 LOC201562 524 201029_s_at 2.06 NM_002414 CD99525 211697_x_at 1.39 AF349314 LOC56902 526 201066_s_at 2.09 NM_001518GTF2I 527 213923_at 1.92 AW005535 RAP2B 528 200024_at 2.1 NM_001009 RPS6529 202378_s_at 1.73 NM_017526 LEPR 530 218856_at 2.13 NM_016629TNFRSF21 531 208816_x_at 2.27 M62898 — 532 201672_s_at 2.1 NM_005151USP14 533 214047_s_at 1.5 AI913365 MBD4 534 212042_x_at 2.12 BG389744RPL7 535 211742_s_at 1.75 BC005926 EVI2B

Modifications and variations of the methods and compositions describedherein will be obvious to those skilled in the art and are intended tocome within the scope of the appended claims. The teachings of thereferences cited herein are specifically incorporated by reference.

1-19. (canceled)
 20. A method of diagnosing the source of localinflammation comprising: determining the expression of one or more genesor proteins encoded thereby in a cell, in a clinical sample obtainedfrom a site of local inflammation, and comparing the expression of thegenes or proteins encoded thereby with their expression in known controlsamples from a comparable site without local inflammation to determineif the site is infected, wherein levels of expression of one or moregenes or proteins encoded thereby, indicative of bacterial infection,from the site of local inflammation are altered by at least two fold bybacterial infection, wherein the sample is selected from the groupconsisting of lymph fluid, cerebrospinal fluid, local fluid, connective,tissue, local tissue, cerebrospinal tissue, and any combination thereof.21. The method of claim 20 wherein the gene expression is detected byexamining nucleic acid expression.
 22. The method of claim 20 whereinthe gene expression is detected by examining protein expression.
 23. Themethod of claim 20 wherein expression of a gene is determined byassaying for an mRNA transcribed from the gene or a protein translatedfrom an mRNA transcribed from the gene.
 24. The method of claim 20wherein the sample comprises neutrophils or other white cells.
 25. Themethod of claim 20 wherein the genes are analyzed on a microarray. 26.The method of claim 20 further comprising comparing the measured levelsof expression or protein with levels from controls obtained fromindividuals with confirmed infection.
 27. The method of claim 20 whereinthe clinical samples and controls are obtained from individuals withconfirmed inflammation due to gout or autoimmune disease.
 28. The methodof claim 20 wherein the expression is compared by comparing proteinexpressed from the genes.
 29. The method of claim 20 wherein the genesare selected from the group consisting of P13, TNFAIP6, GPR43, GBP1,CCL4, CCL3, HM74, MAFF, PLAU, GCH1, SOD2, SLP1, HIG2, IL1RN, FABP5,LIPA, CD36, CD9, GPNMB, FCER1A, FN1, LOC115207, Z39 IG, MAFB, LGMN,CD36, FN1, C6orf62, and MS4A4A.
 30. The method of claim 20 wherein theexpression or protein levels are three fold different.
 31. The method ofclaim 20 wherein the expression or protein levels are five folddifferent.
 32. The method of claim 20 wherein the expression or proteinlevels are ten fold different.
 33. The method of claim 20 wherein theproteins are selected from the group consisting of skin-derivedantileukoproteinase (SKALP) (PI3), interleukin-1beta (IL1B),interleukin-8 (IL8), Interleukin-1 receptor-associated kinase 3 (IRAK3),CC chemokine ligand 3 (CCL3), CC chemokine ligand 4 (CCL4), superoxidedismutase 2 (SOD2), Nuclear Factor of Kappa light polypeptide geneenhancer in B-cells Inhibitor, Alpha (NFKBIA), Nijmegen breakagesyndrome 1 (NBS 1), tumor necrosis factor alpha-induced protein 6(TNFAIP6), and plasminogen activator, urokinase (PLAU).